Academic Writing (Pharmacology)

Here you can view various pieces relevant to pharmacology and toxicology I’ve written throughout my MACPR career:

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Carbon tetrachloride: a novel compound for the research of liver degeneration. 

Several synthetic or naturally occurring chemicals can produce toxic damage within the liver. A quick browse through hepatotoxicology literature undoubtedly reveals that carbon tetrachloride (CCl4) is clearly detrimental to liver functionality. Aside from acetaminophen, carbon tetrachloride is easily one of the most comprehensively studied chemical agents to exert damage on liver parenchyma through known biochemical processes. Relevant damage is easily validated post-administration by observing a variety of degenerative changes in liver morphology. This organic compound does not occur naturally but still lingers throughout the environment. In the past, it was commonly used for manufacturing and cleaning purposes but has since been subjected to stricter regulation because of its known detriment to the liver and other organ systems. Nevertheless, CCl4 continues to dwell in environmental air, water, and soil, making it an important compound for toxicological study. (Fujimoto and Iimuro) The morphological changes and damage in the liver entailing CCl4 administration will be explored at the cellular level followed by a discussion of proposed molecular mechanisms contributing to toxic insult. Lastly, a brief overview of adaptive mechanisms and treatment approaches will be provided.

For decades it has been known that administration of CCl4 to liver cells rapidly induces membrane injury and subsequent loss of permeselectivity. (Recknagel et. al) A significant contribution of the morphological research addressing carbon tetrachloride- induced hepatotoxicity has arisen from utilizing isolated hepatocytes from rodent models. Berger et. al’s research in the late 1980’s outlined a variety of clear morphological changes obtained using light microscopy and transmission electron microscopy methods. The changes appeared to be time-dependent with greater insult occurring at later intervals. Initial changes post-administration include the initial swelling of microvilli tips followed by eventual loss of microvilli altogether, contributing to an overall smooth hepatocyte cell surface. Damage appears to be significantly localized to the endoplasmic reticulum where parallel orientation vanishes, plates scatter throughout the cytoplasm, and ribosomal pruning occurs. The mitochondria exhibit morphological swelling that includes deteriorations in matrix electron density and cristae fragmentation. Chromatin clumping is also evident in the periphery, which is a known sign of cellular injury. (Berger et. al) Irreversible lysosomal damage is also imminent. Profound histological changes typically occur following 5 or more hours of administration and include lobular swelling and an increased presence of cytoplasmic droplets in the form of lipid bodies (Smuckler et. al). Per Christie and Judah, at 18 hours post-administration, mass necrosis is evident around the central vein of the liver and spirals outward with the potential to destroy almost all hepatocytes. (Christie & Judah) These various morphological changes are thought to result from lipid peroxidation in the ER, which is known to be destructive to biological membranes. (Brattin. et al) Further research has also revealed the involvement of Kupffer cells after CCl4 administration. Specifically, CCl4 triggers Kupffer cell activity through a calcium-mediated process, resulting in the release of toxic secretory cytokines that contribute to hepatocyte destruction. (Edwards et. al) Centrilobular hemorrhage follows these inflammatory responses, which ultimately leads to the collapse of centered lobules as well as reticular condensation. (Fujimoto and Iimuro)

It is clear that CCl4 is destroys the liver’s structural integrity. All morphological damage can be explained by molecular and biochemical means. The general process involves reductive dehalogenation of CCl4 resulting in free radicals that then covalently bind to inhibit protein synthesis/assembly and induce lipid peroxidation. (Boll et. al) Specifically, the free radicals involved in these destructive and inhibitory processes include trichloromethyl (CCl3) and trichloromethyl peroxyl (CCl3O2), both of which are formed by a hepatic microsomal enzyme in the liver known as CYP2E1. (Ingawale et. al) The formation of these free radicals may also result from an interaction between CCl4 and an NADPH flavoprotein. (Slater & Sawyer) Reductions in protein synthesis are believed to occur due to these radicals inhibiting nuclear NTPase activity, inhibiting mRNA nucleocytoplasmic transport, and down-regulating genes. (Li et. al) The reactive species also reduce the activity of both glucose-6-phosphotase and NADPH, two compounds that are normally responsible for maintaining glutathione levels to help protect cells from oxidative damage. (Slater & Sawyer) Intoxication has also been linked to the increased circulation of inflammatory cytokines and chemokines including the interleukins, TNF-a, eotaxin 2, and FasL. These inflammatory compounds are capable of inducing profound liver inflammation and are triggered via a sympathetic nervous system-mediated pathway where specific SNS neurotransmitters bind to immune cells gated by purinergic and adrenergic receptors for their release. (Lin et. al) The increased incidence of TNF-a following CCl4 exposure has been repeatedly noted throughout hepatotoxicology literature although no conclusive explanation exists to explain its role in hepatotoxicity. Multiple early studies have paradoxically stated that it may in fact serve as a protective agent but more recent study efforts have deemed it responsible for the development of moderate liver inflammation following chronic CCl4 exposure. (Simeonova et. al) Elevated calcium concentrations appear to be involved in CCl4-induced hepatotoxicity as well. Administration of CCl4 in vivo destroys the Ca2+ sequestering capacity of liver microsomes and the endoplasmic reticulum. This leads to a disruption of regular intracellular Ca2+ concentrations, which is commonly attributed to the disruption of various cellular functions. (Brattin et. al) One such functional disruption includes interference with intracellular traffic of VLDL, which might explain histological observations of lipid deposits. Disruptions also include inhibitory effects on protein synthesis. (Recknagel)

A variety of agents have been postulated to protect liver cells from carbon tetrachloride’s destructive properties. Dietary supplementation of vitamin E can reduce chronic liver disease resulting from consistent CCl4 ingestion by combating membrane damage attributed to lipid peroxidation. Piperonyl butoxide and cytochrome P450 ligand have both proven their ability to abolish all toxic effects of CCl4 in cultured hepatocytes. Piperonyl butoxide can specifically interfere with the covalent binding behavior of CCl4 radicals, releasing the blockage of VLDL and HDL secretions. Ca2+ channel blockers may limit calcium-induced damage to hepatocytes as well. S-adenosyl-methionone can be used to restore depleted glutathione levels for combating oxidizing species. A variety of antioxidant plant products including silymarin and colchicine have been found to protect against CCl4 hepatocyte by inhibiting cytochrome P450. (Boll et. al) It has also been found that the administration of choline by stomach tube in rat models between 0 to 15 hours of CCl4 inhalation can considerably delay the onset of mass necrosis and reduce the overall severity of liver injury in chronic poisoning. (Christie & Judah) Instances of severe or deliberate poisoning should be treated medically by first immediately removing the exposure source. N-acetyl-cysteine (NAC), although normally used for acetaminophen poisoning, may have beneficial effects in limited cases of CCl4 injury. Hyperbaric oxygen has been proven to help combat CCl4 poisoning in both humans and rodents, but only within 6-hours of ingestion. (Fujimoto & Iimuro) The liver’s endogenous approach to combating CCl4 insult includes the simultaneous secretion of TNF-a and nitrous oxide. These two agents work in tandem by down-regulating each other’s production in an effort to reduce cytokine-induced inflammation. (Morio et. al)

Carbon tetrachloride, although a hepatotoxic compound, has made huge contributions to the study of hepatotoxicity. Indeed, breakthroughs in toxicological research have provided solid reasoning to prove that this compound’s use should be limited in order to minimalize toxic insult.

References

Berger, M. L., Reynolds, R. C., & Combes, B. (1987). Carbon tetrachloride-induced morphologic alterations in isolated rat hepatocytes. Experimental and Molecular Pathology, 46(3), 245-257. doi:10.1016/0014-4800(87)90047-5

Boll, M., Lutz, W. D., Becker, E., & Stampfl, A. (2001). Mechanism of Carbon Tetrachloride-Induced Hepatotoxicity. Hepatocellular Damage by Reactive Carbon Tetrachloride Metabolites. Zeitschrift Für Naturforschung C, 56(7-8). doi:10.1515/znc-2001-7-826

Brattin, W. J., Glende, E. A., & Recknagel, R. O. (1985). Pathological mechanisms in carbon tetrachloride hepatotoxicity. Journal of Free Radicals in Biology & Medicine, 1(1), 27-38. doi:10.1016/0748-5514(85)90026-1

Christie, G. S., & Judah, J. D. (1954). Mechanism of Action of Carbon Tetrachloride on Liver Cells. Proceedings of the Royal Society B: Biological Sciences, 142(907), 241-257. doi:10.1098/rspb.1954.0024

Edwards, M. (1993). The Involvement of Kupffer Cells in Carbon Tetrachloride Toxicity. Toxicology and Applied Pharmacology, 119(2), 275-279. doi:10.1006/taap.1993.1069

Fujimoto, J., & Iimuro, Y. (2010). Carbon Tetrachloride-Induced Hepatotoxicity*. Comprehensive Toxicology, 437-455. doi:10.1016/b978-0-08-046884-6.01018-6

Ingawale, D. K., Mandlik, S. K., & Naik, S. R. (2014). Models of hepatotoxicity and the underlying cellular, biochemical and immunological mechanism(s): A critical discussion. Environmental Toxicology and Pharmacology, 37(1), 118-133. doi:10.1016/j.etap.2013.08.015

Li, X. (2010). Mechanism underlying carbon tetrachloride-inhibited protein synthesis in liver. World Journal of Gastroenterology WJG, 16(31), 3950. doi:10.3748/wjg.v16.i31.3950

Lin, J., Peng, Y., Wang, S., Young, T., Salter, D. M., & Lee, H. (2015). Role of the Sympathetic Nervous System in Carbon Tetrachloride-Induced Hepatotoxicity and Systemic Inflammation. PLOS ONE PLoS ONE, 10(3). doi:10.1371/journal.pone.0121365

Morio, L. A., Chiu, H., Sprowles, K. A., Zhou, P., Heck, D. E., Gordon, M. K., & Laskin, D. L. (2001). Distinct Roles of Tumor Necrosis Factor-α and Nitric Oxide in Acute Liver Injury Induced by Carbon Tetrachloride in Mice. Toxicology and Applied Pharmacology, 172(1), 44-51. doi:10.1006/taap.2000.9133

Recknagel, R. O., Glende, E. A., & Hruszkewycz, A. M. (1977). Chemical Mechanisms in Carbon Tetrachloride Toxicity. Free Radicals in Biology, 97-132. doi:10.1016/b978-0-12-566503-2.50010-0

Recknagel, R. O. (1983). A new direction in the study of carbon tetrachloride hepatotoxicity. Life Sciences, 33(5), 401-408. doi:10.1016/0024-3205(83)907877

Recknagel, R. O. (1983). Carbon tetrachloride hepatotoxicity:status quo and future prospects. Trends in Pharmacological Sciences, 4, 129-131. doi:10.1016/0165- 6147(83)90328-0

Simeonova, P. P., Gallucci, R. M., Hulderman, T., Wilson, R., Kommineni, C., Rao, M., & Luster, M. I. (2001). The Role of Tumor Necrosis Factor-α in Liver Toxicity, Inflammation, and Fibrosis Induced by Carbon Tetrachloride. Toxicology and Applied Pharmacology, 177(2), 112-120. doi:10.1006/taap.2001.9304

Slater, T., & Sawyer, B. (1970). The hepatotoxic action of carbon tetrachloride stimulatory effect of carbon tetrachloride on lipid peroxidation in microsomal suspensions. FEBS Letters, 11(2), 132-136. doi:10.1016/0014- 5793(70)80510-5

Smuckler, E. A. (1962). An Intracellular Defect In Protein Synthesis Induced By Carbon Tetrachloride. Journal of Experimental Medicine, 116(1), 55-72. doi:10.1084/jem.116.1.55

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The nephrotoxic potential associated with lithium treatment and overdose. 

It is not uncommon in modern medicine for there to be instances where a drug prescribed for the treatment of one organ system ends up interfering with the functionality of another. Such is the case for lithium. Lithium administration has been linked to clear functional changes in nervous system activity that can help attenuate a variety of behavioral abnormalities implicated in psychiatric diseases such as suicidal tendencies, manic depression, and bipolar behavior. As such, this drug’s favorable psychiatric profile makes it a widely prescribed agent for numerous behavioral conditions. Despite its known therapeutic legitimacy, however, lithium has also been linked to functional and morphological damage to the kidneys. The molecular mechanism(s) entailing lithium’s nephrotoxic properties are inherently complex and far from understood but have nonetheless been repeatedly examined in the literature. Through the analysis of several peer-reviewed publications, multiple hypotheses explaining the mechanistic action of lithium-induced nephrotoxicity will be discussed. Then, observed morphological damages resulting from its toxic insult will be addressed followed by a brief overview of current treatment and prevention measures.

Lithium administration negatively impacts the kidneys at the level of the nephron where concentrations tend to plateau within 12 to 24 hours after administration. Lithium is capable of maneuvering across semi-permeable membranes with ease and is freely filtered through the glomerulus, with 80% being reabsorbed in the proximal convoluted tubule. (Chan et. al) Acute treatment leads to reductions in vasopressin (ADH) concentrations as well as weakening in vasopressin strength. Such ADH deficits are problematic given that this hormone is known to produce adenylate cyclase. Adenylate cyclase is an important agent actively involved in the production of cyclic-AMP, which is used to regulate the normal flow of water. (Chan et. al) Abnormal serum creatinine levels are typically observed following lithium treatment, which can be indicative of GFR impairment. The magnitude of these renal deficits is linked to treatment duration. Indeed, patients undergoing longer treatment windows show significantly elevated levels of creatinine and urea. It is important to acknowledge, however, that reduced GFR can still occur in the initial stages of lithium therapy as well. (Azab et. al) Other commonly reported renal complications include polyuria, polydipsia, and impairment of renal concentration processes. (Chan et. al) Polyuria likely occurs due to collecting duct resistance to ADH following lithium administration. (Coskunol et. al) de Oliveira and colleagues have linked lithium treatment to a decreased density of AVP receptors used for ADH binding as well as to reduced expression of AQP2, a protein-coding gene used for aquaporin production. They have also proposed a linkage between lithium and COX- 2 expression in the kidney medulla, which is linked to prostaglandin synthesis that further elevates polyuric dilution (de Oliveira et. al) Grunfeld & Rossier have further elaborated upon these findings by proving that AQP2 dysregulation is accompanied by concomitant dysregulation of epithelial sodium channel (ENaC) expression in the cortical and medullary collecting ducts. (Grunfeld & Rossier) McKnight et. al have also added to the AQP2 hypothesis by suggesting that this dysregulation is linked lithium’s inhibition of a G-protein coupled pathway. (McKnight et. al) Despite the variability of these mechanistic explanations, all findings ultimately converge upon nephrogenic diabetes insipidus (NDI) as a result, which the most common renal side effect accompanying lithium therapy. (Markowitz. et. al) Additional reported effects include the modified excretion of electrolytes including sodium, potassium, and phosphate as well as alterations in salt and water likely resulting from previously mentioned inhibitory ADH mechanisms. (Myers et. al.) Lastly, Tredget and colleagues have suggested that lithium treatment may progress beyond NDI and lead to end-stage renal disease (ESRD). These findings, although largely unsubstantiated, have been proven in small population samples in a few studies. Tredget’s team drew upon this hypothesis by mentioning a male manic- depressive patient who developed ESRD 12 years after lithium treatment initiation. While the team claimed ESRD to be a possible consequence, they acknowledged it to be a rare event that only occurs within a small portion of patients with abnormally high eGFR reductions. (Tredget et. al)

Much of the morphological damage associated with lithium treatment has been analyzed through renal biopsy studies. These studies reveal clear histopathological changes after long-term administration characterized by interstitial fibrosis, tubular atrophy, and glomerular sclerosis. (Hetmar et. al) Interestingly, tubular damage is likely linked to previously mentioned impairments in urinary concentration. Renal lesions often accompany lithium-induced tubular damages. MRI studies have identified bilateral renal microcysts that uniformly distribute throughout both the cortex and the medulla (Neugarten & Golestaneh) Within the context of the previously mentioned AQP2 dysregulation, reduced gene expression is histopathologically manifested in three ways: altered cellular organization within the collecting ducts, an increase in intercalated cells, and a reduction of principal cells. (Walker & Endre) Patient biopsies also illustrate further lesion damage to the collecting ducts as well as the distal convoluted tubules Walker et. al have dubbed these types of lesions ‘acute specific tubular lesions’, which are typically characterized by swelling and vacuolization of the cytoplasm. (Walker et. al) Electron microscopy has defined interstitial fibrosis-induced cellular damage by the presence of swollen cells, elevated mitochondrial concentrations, damaged endoplasmic reticula, vacuolation of apical membranes, and the aggregation of glycogen granules in the cytoplasm. (Walker & Endre) Per Grunfeld and Rossier, such interstitial fibrosis can appear as early as 5 years after the initiation of lithium therapy. (Grunfeld and Rossier) Glomerusclerosis is another possibility; Markowitz and colleagues noted glomerular hardening in 50% of renal biopsy samples of 24 patients enrolled in long-term lithium treatment. (Markwoitz et. al) Hetmar et. al have contributed additional quantitative data by demonstrating that patients under lithium treatment show an alarming five times as many sclerosed glomeruli, three times as many atrophic tubules, and twice as much fibrosis in comparison to control patients of the same age group. (Hetmar et. al) Indeed, these various findings make it clear that the nephrotoxic effects of lithium treatment can compromise the efficacy of the kidney’s cellular structure.

Both the psychiatrist and nephrologist should closely monitor any patient undergoing lithium therapy. Kidney health must be thoroughly assessed before initiating treatment. Kidney function should be evaluated biannually through the analysis of GFR, eGFR, or 24-hour creatinine clearance readings. (Severus & Bauer) Currently, the most pharmacologically established treatment for lithium-induced NDI is amiloride administration. Treatment efficacy for amiloride correlates with the severity of kidney damage with lesser damage yielding to higher success. (Azab et. al) Amiloride functions by blocking sodium entry via the ENaC and inhibiting lithium entry into the collecting

duct principal cells. It also might be linked to an elevated expression of AQP2. (de Oliveira et. al) Neugarten & Golestaneh suggest combating volumetric depletion through fluid resuscitation procedures, enteric lavage for the prevention of further lithium absorption, administration of polystyrene sulfonate, and renal replacement therapy if necessary. (Neugarten & Golestaneh) Acute lithium intoxication can also be managed through hemodialysis procedures. (Markowitz et. al) Within the context of ESRD, although information is still lacking, Tredget et. al suggest halting lithium early as an ideal prevention mechanism. (Tredget et. al)

Ultimately it is clear that there is some risk to kidney health when lithium treatment is administered. However, the risks appear to be largely time and dose- dependent and are thusly avoidable if the attending physician remains vigilant in monitoring kidney health. Strict dosing and regular screening will minimalize the concern of functional and morphological damage when treating behavioral conditions with lithium drugs.

References

Azab, A. N., Shnaider, A., Osher, Y., Wang, D., Bersudsky, Y., & Belmaker, R. H. ( 2015). Lithium nephrotoxicity. International Journal of Bipolar Disorders, 3, 13. http://doi.org/10.1186/s40345-015-0028-y

Chan WY, Mosca P, Rennert OM. Lithium nephrotoxicity: a review. Ann Clin Lab Sci. 1981 Jul-Aug;11(4):343-9. Review. PubMed PMID: 7023348.

Coşkunol, H., Vahip, S., Mees, E. D., Başçi, A., Bayindir, O., & Tuğlular, I. (1997). Renal side-effects of long-term lithium treatment. Journal of Affective Disorders, 43(1), 5-10. doi:10.1016/s0165-0327(96)00046-8

Grünfeld, J., & Rossier, B. C. (2009). Lithium nephrotoxicity revisited. Nat Rev Nephrol Nature Reviews Nephrology, 5(5), 270-276. doi:10.1038/nrneph.2009.43

Hetmar, O., Brun, C., Ladefoged, J., Larsen, S., & Bolwig, T. G. (1989). Long-term effects of lithium on the kidney: Functional-morphological correlations. Journal of Psychiatric Research, 23(3-4), 285-297. doi:10.1016/0022-
3956(89)90034-4

Markowitz GS, Radhakrishnan J, Kambham N, Valeri AM, Hines WH, D’Agati VD. Lithium nephrotoxicity: a progressive combined glomerular and tubulointerstitial nephropathy. J Am Soc Nephrol. 2000 Aug;11(8):1439-48. PubMed PMID: 10906157.

Mcknight, R. F., Adida, M., Budge, K., Stockton, S., Goodwin, G. M., & Geddes, J. R. (2012). Lithium toxicity profile: A systematic review and meta-analysis. The Lancet, 379(9817), 721-728. doi:10.1016/s0140-6736(11)61516-x

Myers, J. B., Morgan, T. O., Carney, S. L., & Ray, C. (1980). Effects of lithium on the kidney. Kidney International, 18(5), 601-608. doi:10.1038/ki.1980.178

Neugarten, J., & Golestaneh, L. (2014). Nephrotoxicity of Lithium and Drugs of Abuse☆. Reference Module in Biomedical Sciences. doi:10.1016/b978-0-12- 801238-3.02057-2

Oliveira JL, Silva Júnior GB, Abreu KL, Rocha Nde A, Franco LF, Araújo SM, Daher Ede F. Lithium nephrotoxicity. Rev Assoc Med Bras. 2010 Sep-Oct;56(5):600-6. Review. English, Portuguese. PubMed PMID: 21152836.

Severus, E., & Bauer, M. (2013). Managing the risk of lithium-induced nephropathy in the long-term treatment of patients with recurrent affective disorders. BMC Medicine BMC Med, 11(1), 34. doi:10.1186/1741-7015-11-34

Tredget, J., Kirov, A., & Kirov, G. (2010). Effects of chronic lithium treatment on renal function. Journal of Affective Disorders, 126(3), 436-440. doi:10.1016/j.jad.2010.04.018

Walker, R. J., & Endre, Z. H. (2008). Cellular Mechanisms of Drug Nephrotoxicity. Seldin and Giebisch’s The Kidney, 2507-2535. doi:10.1016/b978-012088488- 9.50090-5

Walker, R. G., Davies, B. M., Holwill, B. J., Dowling, J. P., & Kincaid-Smith, P. (1982). A clinico-pathological study of lithium nephrotoxicity. Journal of Chronic Diseases, 35(8), 685-695. doi:10.1016/0021-9681(82)90021-2

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Obstructive effects of ozone on the pulmonary system. 

Respiration is a delicate albeit crucial physiological process mediated by the body’s intricate pulmonary system. As is the case in other systems, this process can be subject to disruption after toxicant exposure. Respiratory toxicology stems from the inhalation of toxic agents that exert their effects on the lung, the major organ responsible for facilitating respiratory function. While the lung’s remarkably large surface area optimizes its ability to engage in gas exchange, this structural feature comes with a price: a heightened vulnerability to damage at the parenchymal, bronchiole, and alveolar level. Indeed, a host of agents in the toxicological literature have been found to contribute to respiratory dysfunction at various levels of understanding. One of the most comprehensively studied agents is ozone, an oxygenic gas with toxic potential that has been evaluated through evidence-based research for several decades.

Ozone is a highly reactive gaseous environmental pollutant produced by atmospheric chemical reactions and is known to contribute to respiratory illness. Though it has been studied comprehensively, the exact mechanisms behind its respiratory toxicity have yet to be fully understood. Nonetheless, epidemiological data have clearly revealed a correlation between elevated ozone exposure and increased hospital admission for respiratory distress. (Clay et. al) This clinical significance has sparked the interest of pulmonary researchers and toxicologists alike in investigating its molecular and biochemical modes of action. Molecular findings generally converge on the principle that ozone exposure causes a variety of inflammatory-mediated cellular processes. One body of research postulates that ozone induces lung inflammation through neutrophil infiltration and the release of inflammatory cytokines such as TNFα and various interleukins. The two most influential interleukins appear to be IL-1ß and IL-18, which are controlled by an inflammatory signaling pathway dubbed by Che et. al as the ‘inflammasome’. This pathway arises from ozone’s strong oxidizing properties, which trigger the mitochondrial release of ROS in lung macrophages that in turn induce the secretion of IL-1ß. (Che et. al) Ozone’s impact on macrophage activity has also been evaluated on other principles aside from just IL-1ß secretion. Interestingly, it has been found that ozone is directly cytotoxic to macrophages, impeding their ability to engage in phagocytosis, intracellular killing, and the secretion of protective factors. (Hollingsworth et. al) Research at the genetic level has linked ozone toxicity to altered microRNA and DNA expression in the lung. Epithelial cell culture methods have demonstrated ozone to trigger the release of IL-6, which then targets microRNA to negatively impact protein expression in the human bronchial epithelial cell line. (Clay et. al) Similarly, ozone acts as an initiator in the production of secondary toxic agents to negatively impact DNA structure. These secondary agents include lipid peroxidative products that generate ROS, which directly damage DNA. For example, these ROS agents can cause the formation of a well studied DNA adduct known as 8-oxoguanine that is known to cause mutagenic G- A transversion effects. (Kosmider et. al) Numerous other molecular mechanisms exist to explain ozone’s negative impact on respiratory function, illustrating that its effects on respiratory efficacy are indeed complicated and profound.

Ozone’s toxic properties appear to be obstructive rather than restrictive. Data exists to validate this classification and is best analyzed by observing ozone’s impact on respiratory function at the organ-wide level. Laboratory studies have validated that ozone can negatively affect the permeability of the lung epithelium, creating a disruptive barrier-like environment that is further aggregated by elevated mucosal production. (Bhalla) Adverse lung function has also been experimentally evaluated in several populations with varying ozone concentrations to determine its magnitude of toxicity. It appears, through the scrutiny of several studies, that adverse lung function is consistently present when ozone exposure is greater than or equal to 80 ppb. Concentrations at this level lead to vagal stimulation that impacts pulmonary functionality by inducing bronchoconstriction, a phenomenon that is characteristic of a variety of obstructive lung diseases and elicits clear decrements in FEV1 and FVC. Additionally, these higher concentrations have the capacity to overwhelm the lungs’ antioxidant defense system, which regularly provides protection to airway epithelial cells. However, it is important to note that these elevated concentrations are experimentally induced and not indicative of regular atmospheric exposure (Goodman et. al) In a more practical approach, Kim et. al observed the lung inflammatory response in young adults exposed to 0.06 ppm of ozone for 6.6 hours to evaluate its impact on respiratory function. It was concluded through the analysis of sputum samples that ozone exposure at this interval can certainly cause pulmonary inflammation by altering the airway environment. (Kim et. al) Clear morphological damage is also visible after ozone injury to the respiratory tract. For example, ozone sloughs ciliated cells in the upper and lower conducting airways that normally work to keep them clean for healthy breathing. Another area found to be specifically susceptible to damage is the pulmonary acinus, which contains terminal bronchioles and alveolar ducts involved in gas exchange. Within the conducting airways, alveolar type I cells undergo damage although type II cells are capable of surviving ozone-induced injury. Additionally, exposure is also postulated to induce hyperplasia commonly found in lung cancer, suggesting it may be a potential carcinogen. (Mustafa)

Atkinson and colleagues observed the relationship between long-term exposure to ambient ozone and mortality in an evidence-based meta-analytic study. The researchers concluded that there is great evidence to link short-term ozone exposure to a variety of adverse health outcomes. Although these findings have been consistently reinforced in the literature, the research team was more so interested in gathering data with respect to ozone’s long-term respiratory implications since they have not been studied comprehensively. Through the evaluation of 14 relevant publications released between September and October 2015, no evidence was found to associate long-term annual ozone concentrations with the risk of death from respiratory disease. (Atkinson et. al) Indeed, it is difficult to draw concise conclusions about the effect of chronic ozone inhalation on the lungs since most experimental studies provide data on toxicity of short- term, highly concentrated exposures. However, it is postulated that there may be an acceleration of lung function deterioration that could ultimately lead to various obstructive lung diseases such as chronic bronchitis or asthma. (Mustafa)

Plenty of evidence exists to demonstrate that ozone is in fact a respiratory toxicant. Several research findings detail its potential cellular and molecular modes of action as well as its impact on lung functionality by means of inflammation and obstruction of the bronchioles and alveoli. Based on these current short-term findings, it is likely safe to conclude that ozone is a dangerously toxic respiratory agent that requires consistent study. However, it will be important to develop a greater understanding of the long-term implications associated with regular inhalation at lower concentrations, as this is more reflective of ozone’s atmospheric abundance in our daily environment. Such studies would provide great insight on the prevention of respiratory toxicity.

References

Atkinson, R. W., Butland, B. K., Dimitroulopoulou, C., Heal, M. R., Stedman, J. R., Carslaw, N., . . . Anderson, H. R. (2016). Long-term exposure to ambient ozone and mortality: A quantitative systematic review and meta-analysis of
evidence from cohort studies. BMJ Open, 6(2).

Bhalla, D. K. (1999). Ozone-Induced Lung Inflammation And Mucosal Barrier Disruption: Toxicology, Mechanisms, And Implications. Journal of Toxicology and Environmental Health, Part B, 2(1), 31-86.

Che, L., Jin, Y., Zhang, C., Lai, T., Zhou, H., Xia, L., . . . Shen, H. (2016). Ozone- induced IL-17A and neutrophilic airway inflammation is orchestrated by the caspase-1-IL-1 cascade. Sci. Rep. Scientific Reports, 6, 18680.

Clay, C. C., Maniar-Hew, K., Gerriets, J. E., Wang, T. T., Postlethwait, E. M., Evans, M. J., . . . Miller, L. A. (2014). Early Life Ozone Exposure Results in Dysregulated Innate Immune Function and Altered microRNA Expression in Airway Epithelium. PLoS ONE, 9(3).

Goodman, J. E., Prueitt, R. L., Chandalia, J., & Sax, S. N. (2013). Evaluation of adverse human lung function effects in controlled ozone exposure studies. J. Appl. Toxicol. Journal of Applied Toxicology, 34(5), 516-524.

Hollingsworth, J. W., Kleeberger, S. R., & Foster, W. M. (2007). Ozone and Pulmonary Innate Immunity. Proceedings of the American Thoracic Society, 4(3), 240- 246.

Kim, C. S., Alexis, N. E., Rappold, A. G., Kehrl, H., Hazucha, M. J., Lay, J. C., . . . Diaz- Sanchez, D. (2011). Lung Function and Inflammatory Responses in Healthy Young Adults Exposed to 0.06 ppm Ozone for 6.6 Hours. Am J Respir Crit
Care Med American Journal of Respiratory and Critical Care Medicine, 183(9), 1215-1221.

Kosmider, B., Loader, J. E., Murphy, R. C., & Mason, R. J. (2010). Apoptosis induced by ozone and oxysterols in human alveolar epithelial cells. Free Radical Biology
and Medicine, 48(11), 1513-1524.

Mustafa, M. G. (1990). Biochemical basis of ozone toxicity. Free Radical Biology and Medicine, 9(3), 245-265.

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Acetaminophen hepatotoxicity: morphological degeneration, biochemical pathways, and treatment methods

Tylenol is the most commonly used fever reducer and mild pain reliever in the United States. The active ingredient is acetaminophen (also known as paracetamol), a compound comprised of a benzene ring core modified by the substitution of one hydroxyl group and an amide group. Though utilized for several years, the analgesic mechanism of action behind acetaminophen is still not fully understood. Nonetheless, it is consistently used for the alleviation of generalized pain by millions of consumers yearly. The general consensus of the medical community is that adherence to dosage labeling makes it a safe drug of choice to all healthy individuals and even some individuals suffering from illness. However, it is important to note that this drug is also an excellent example of how a therapeutic agent can have toxic implications when taken irresponsibly. It has long been established that acetaminophen can cause hepatic damage in a dose-dependent fashion and current research continues to reveal new information regarding the mechanistic action of these hepatotoxic effects. In order to grasp past and current research findings, a brief discussion of liver physiology and morphology relating to the actions of acetaminophen will be presented followed by a variety of proposed hypotheses in the literature on how it exerts its hepatotoxic damage at the metabolic and molecular level. Lastly, endogenous adaptive mechanisms and synthetic treatment approaches following toxic insult will be briefly addressed.

Acting as the body’s largest organ, the liver is a complex morphological structure responsible for the majority of our internal detoxification processes. It is comprised of a variety of cells that collectively facilitate an elaborate mechanism of toxicant extraction from the blood. Liver physiology is complicated and goes well beyond the extent of just acetaminophen toxicity. Thus, for the scope of this topic, only structures relevant to the proposed toxic insult will be addressed. Acetaminophen is readily absorbed from the gastrointestinal tract into the blood. (Bari & Fontana) In cases of overdose, high blood concentrations can lead to acute liver failure (ALF) that may cause impairments in bilirubin elimination as well as complex coagulopathy. Cellular destruction is also very much a possibility depending on the concentration of acetaminophen ingested. This destructive mechanism primarily involves a characteristic pattern of pericentral necrosis resulting from a well-studied metabolic toxicity mechanism that will be later discussed. (Fontana) Indeed, necrosis of hepatocytes, the liver’s main parenchymal cells, can be fatal due to their widespread roles in protein synthesis and storage, bile salt synthesis, and detoxification. This necrosis is thought not only to stem from metabolic dysfunction but also from ATP depletion. (Hinson et. al) As early as 1966, Davidson and Eastham were able to characterize the morphological detriment accompanying acetaminophen overdose through histological study. Microscopic examinations of liver sections from deceased overdosing individuals reveal clear necrotic activity in the centrilobular zone followed by eosinophilic degeneration of damaged hepatic cells. There are also clear signs of early degeneration and vacuolization in portal sites peripheral to the centrilobular area. (Davidson and Eastham) Oncotic necrosis has also has been identified via TUNEL staining of hepatic sections in mice. The stained hepatic sections reveal chromatin clumping within hepatic nuclei as well as cytoplasmic clumping around the centrilobular zones. (Ishii et. al) Morphological studies have also revealed that congestion can occur following toxic acetaminophen doses due to the potential accumulation of red blood cells in the Space of Disse, the persinusoidal region between hepatocytes and sinusoids. This can lead to a subsequent collapse of the sinusoidal lumen, an important structure that houses Kupffer cells (resident macrophages) and is responsible for receiving blood from the portal vein. (Walker et. al) Plasma membrane impairment is also evident following the administration of a toxic acetaminophen dose of 1500 mg/kg in mice. The resultant liver damage compromises the transport functionality of the hepatocytes, thus damaging the membrane and increasing enzymatic activity. This leads to further cellular leakage and cellular dysfunction in a positive feedback-like fashion. (Ilavenil et. al) Lastly, few research efforts have suggested the possibility of apoptosis as another mechanism contributing to acetaminophen-induced hepatocyte damage although these claims are lacking. Even still, the apoptotic theory has been validated through the examination of liver cells after acetaminophen-induced cell death in patients where a low percentage of damaged cells appear to demonstrate hallmark apoptotic properties including cell shrinkage, chromatin condensation, and significant caspase activation. However, the low proportion of these cells makes this hypothesis inadequate in comparison to the general consensus of necrotic mechanisms suggested by the bulk of the medical community. (McGill et. al)

Several molecular mechanisms have been proposed to contribute to the hepatic toxicity of acetaminophen. However, one specific metabolic mode of action tends to recur throughout the literature, which explains that hepatic necrosis is induced through a cytochrome P-450-mediated oxidative process. (Fontana et. al) At normal doses, acetaminophen is converted by P-450 into a reactive toxicant known as N-acetyl-p- benzo-quinone imine (NADPQI). At regular therapeutic doses, the body is capable of detoxifying this reactive intermediate using the glutathione (GSH) system, where glutathione binds to form a harmless acetaminophen-glutathione conjugate. However, at higher (toxic) doses of acetaminophen, the GSH system becomes overwhelmed leading to GSH depletion and a buildup of toxic NADPQI. This NADPQI then covalently bonds to critical proteins within the liver leading to structural degradation. (Gibson et. al) Sidney Nelson has suggested that these specific proteins are likely mitochondrial proteins that are regularly responsible for energy production as well as proteins involved in cellular control (Nelson). Corcoran and colleagues have further elaborated upon this mechanism by suggesting that GSH depletion and gross covalent bonding can also lead to impairment of intracellular calcium regulation. This is because GSH depletion is thought to change activity of phosphorylase a, an enzyme used for the hepatic maintenance of intracellular calcium. Damage to this calcium control mechanism is believed to lead to calcium buildup, which is an early, critical event of drug-induced cell death. (Corcoran et. al) Several other research efforts have speculated that hepatic necrosis can result from oxidative stress. A variety of mechanisms utilizing neutrophils, CYP2E1, nitric oxide, and more exist to justify this proposal although for the sake basic explanation only one hypothesis will be briefly discussed. Normally, GSH is responsible for detoxifying peroxynitrite, an oxidating agent. However, the NADPQI depletion of GSH causes peroxynitrite to nitrate tyrosine, forming a unique 3-nitrotryosine compound that is a unique biomarker of nitrogen stress. Clear deposits of this nitrated tyrosine variant have been found in mice treated with toxic doses of acetaminophen in hepatic proteins of the centrilobular regions that cause protein adducts similar to those of NADPQI. (Hinson et. al) Lastly, acetaminophen toxicity may be linked to a loss of mitochondrial functionality. Toxic overdose leads to increases of mitochondrial calcium, mitochondrial swelling, and the release of intramitachondrial ions and reactive oxygen and nitrogen species. (James et. al)

Diagnosis of overdose is obtained through initial serum measurements of acetaminophen using alanine aminotransferase (ALT) and aspartate aminotransferase (AST). (Fontana) Within 4 hours of acetaminophen overdose, standard medical practice involves the administration of ipecac syrup to expunge the toxicant via vomiting as well as through gastric lavage. Activated charcoal may then be used to reduce absorption of all remaining internal toxicant. (Bari & Fontana) Magnesium and sodium sulfate cathartics may also be useful in enhancing acetaminophen sulfation although more efficacy research is required to validate this claim. (Jackson et. al) Standard practice within 24- hours of overdose also entails the administration of N-acetyl-cysteine, which is used to restore intracellular GSH levels following overdose and has an estimated success rate of 66%. Severely poor prognosis of irreversible damage requires emergency liver transplantation to ensure any chance of survival. (Fontana) The liver itself can also employ a variety of adaptive mechanisms to combat toxicant insult. Such mechanisms include the proliferation of mature hepatocytes, secretion of TNF-a and IL-6 to provide additional pro-mitotic effects during the proliferation process, and the release of vascular endothelial growth factor (VEGF) to improve interaction between endothelial cells and hepatocytes and possibly improve hepatic blood flow. (Hinson et. al) Indeed, while acetaminophen toxicity can have drastic impacts on the health and efficacy of the liver, there are a variety of medical and endogenous procedures to minimalize damage and treatment mechanisms are continuing to advance. However, adherence to dosage labeling can help alleviate concerns altogether through easy prevention rather than treatment.

References

Bari, K., & Fontana, R. J. (2014). Acetaminophen overdose: What practitioners need to know. Clinical Liver Disease, 4(1), 17-21.

Corcoran, G. (1988). Immediate rise in intracellular calcium and glycogen phosphorylase a activities upon acetaminophen covalent binding leading to hepatotoxicity in mice. Toxicology, 50(2), 157-167.

Davidson, D. G., & Eastham, W. N. (1966). Acute liver necrosis following overdose of paracetamol. Bmj, 2(5512), 497-499.

Fontana, R. J. (2008). Acute Liver Failure Including Acetaminophen Overdose. Medical Clinics of North America, 92(4), 761-794.

Gibson, J. D., Pumford, N. R., Samokyszyn, V. M., & Hinson, J. A. (1996). Mechanism of Acetaminophen-Induced Hepatotoxicity: Covalent Binding versus Oxidative Stress. Chem. Res. Toxicol. Chemical Research in Toxicology, 9(3), 580-585.

Hinson, J. A., Roberts, D. W., & James, L. P. (2009). Mechanisms of Acetaminophen- Induced Liver Necrosis. Handbook of Experimental Pharmacology Adverse Drug Reactions, 369-405.

Ilavenil, S., Al-Dhabi, N., Srigopalram, S., Kim, Y. O., Agastian, P., Baru, R., . . . Arasu, M. V. (2016). Acetaminophen Induced Hepatotoxicity in Wistar Rats—A Proteomic Approach. Molecules, 21(2), 161.

Ishii, I., Kamata, S., Hagiya, Y., Abiko, Y., Kasahara, T., & Kumagai, Y. (2015). Protective effects of hydrogen sulfide anions against acetaminophen-induced hepatotoxicity in mice. J. Toxicol. Sci. The Journal of Toxicological Sciences, 40(6), 837-841.

Jackson, C. H., MacDonald, N. C., & Cornett, J. W. (1984). Acetaminophen: a practical pharmacologic overview. Canadian Medical Association Journal, 131(1), 25–37.

James, L. P. (2003). Acetaminophen-Induced Hepatotoxicity. Drug Metabolism and Disposition, 31(12), 1499-1506.

Mcgill, M. R., Sharpe, M. R., Williams, C. D., Taha, M., Curry, S. C., & Jaeschke, H. (2012). The mechanism underlying acetaminophen-induced hepatotoxicity in humans and mice involves mitochondrial damage and nuclear DNA fragmentation. Journal of Clinical Investigation J. Clin. Invest., 122(4), 1574- 1583.

Nelson, S. (1990). Molecular Mechanisms of the Hepatotoxicity Caused by Acetaminophen. Semin Liver Dis Seminars in Liver Disease, 10(04), 267-278.

Walker, R. M., Racz, W. J., & Mcelligott, T. F. (1985). Acetaminophen-Induced hepatotoxic congestion in mice. Hepatology, 5(2), 233-240.

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Nitrous oxide-induced neurodegeneration: a brief overlook. 

It is not uncommon in medical practice to use chemical agents for therapeutic purposes despite their potentially toxic implications. Often times there is no known alternative and questionable drugs are used if benefit is perceived to exceed risk. This concept holds especially true with respect to anesthesiology. There are plenty of procedures that require varying levels of anesthetic to ensure that the patient is not suffering from overwhelming pain or a liability to treatment success. Indeed, a variety of anesthetics within the literature appear to harbor extensive toxicity profiles to various organ systems. One of particular relevance to the nervous system is nitrous oxide. Over five decades of peer-reviewed research exists to suggest that nitrous oxide can be detrimental to neuronal health and survival. The nature of this compound will be discussed in context of its impact on organ system function, metabolism, biochemical interaction, and dosing.

Nitrous oxide is a colorless gaseous compound used in medical practice for analgesia and anesthetization as well as in dangerous recreational drug behavior. It is often referred to as ‘laughing gas’ as it can induce feelings of pleasure when inhaled. The mechanistic action underlying its analgesic effects is well understood but information regarding its anesthesia, while existent, is still lacking. Nonetheless, it is postulated to induce analgesia through opioid mechanisms to elicit antinociceptive properties at the spinal level. Generally speaking, nitrous oxide binds to opioid κ receptors to stimulate the release of endogenous opioid dynorphin peptides to inhibit pain impulses that would normally arise from nociceptive nerve fibers. However, the exact locations of these opioid κ receptors on the nerve terminals is not yet known, nor are the specific locations involved in dynorphin release. With respect to anesthesia, a current hypothesis exists to explain nitrous oxide’s effects through the involvement of ligand-gated ion channels. Specifically, it is believed to induce anesthesia by binding to a combination of receptor channels sensitive to GABA, glycine, nicotinic acetylcholine, 5-hydroxytryptamine3, and glutamate, with the GABAA receptor likely being the most influential. (Emmanouil & Quock) Note, however, that these are just some of many mechanisms proposed for nitrous oxide’s mode(s) of action. Given the vast array of proposed hypotheses, it is clear that the mechanisms behind nitrous oxide’s therapeutic effects are indeed pharmacologically complex.

Within the realm of toxicology, nitrous oxide’s detrimental effects can be observed at the molecular level of the nervous system. The associated metabolic processes and biochemical interactions are complicated but well understood given that interest in nitrous oxide’s chemical action began to peak by the mid-1950’s as neurological complications became increasingly documented. The process is multi- stepped and involves basic principles of redox. Nitrous oxide enters the body after inhalation and binds irreversibly to cobalt in vitamin B12, oxidizing the cobalt to disable vitamin B12 activity. The deactivation of vitamin B12 is especially problematic for the nervous system (especially during developmental stages) because vitamin B12 is a critical remethylating agent to an enzyme known as methionine synthetase, which is used to dampen the effects of homocysteine and contribute to DNA synthesis and repair. In fact, it bas been shown in rodent models that nitrous oxide can inhibit activity of this enzyme by up to 50% just 4 hours after administration. These associated complications are manifested as retardations in neuronal development and elevated release of homocysteine, which can cause damage to important neuroprotective brain microvascular endothelial cells. (Schmitt & Baum) Nitrous oxide is also thought to induce neuronal apoptotic behavior using a mitochondrial pathway through the inhibition of respiratory function. This, of course, can significantly compromise neuronal survival given the level of dependence the nervous system has on energy production via cellular respiration. Nitrous oxide is also linked to an increase of intracellular calcium within the mitochondria, which may contribute to excitotoxicity and consequential neuronal destruction. (Brüne)

The molecular abnormalities can be further validated by observing the brain at the organ-wide level. In one study, Rizzi et. al evaluated the effects of isofluorane and nitrous oxide anesthetics on the brains of fetal guinea pigs. Administration of these drugs revealed significant apoptotic damage to neurons within the cortex, specifically within cingulate cortical layer II. This neuroapoptotic damage was found to increase up to 25- fold when both compounds were administered synergistically. Thus, it is clear that specific cortical regions are very much vulnerable to reduction of neuronal density after anesthetic treatment. (Rizzi et. al) Nitrous oxide has also been found to negatively impact developmental synaptogenesis in rodent pups. Normally within the developmental phase, there is a moderate level of neuronal apoptotic pruning as new connections are formed simultaneously. However, it is speculated that nitrous oxide distorts the regular timing mechanism for pruning during the developmental phase, resulting in apoptotic activity that is instead harmful to neuronal development. (Rooks) Nitrous oxide anesthetic treatment has also been linked to increases of amyloid beta concentrations within the brain. When administered in a 70% nitrous oxide plus 1% isofluorane anesthetic complex, amyloid beta levels elevate, which can further aggregate neuronal apoptosis in a positive feedback mechanism. This alarming finding emulates the pathogenesis and associated neurotoxicity of Alzheimer’s disease. (Zhen et. al)

Strategies exist to minimize toxicity during anesthetic treatment. Abstaining from the use of anesthesia altogether is not a practical approach since the ultimate goal of medicine is to avoid patient harm. Thus, the benefit of pain inhibition almost certainly outweighs the stress and discomfort that any patient would suffer from if certain treatments were to be carried out without anesthesia. Savage and Ma suggest that the most merited solutions involve either supplementing nitrous oxide with neuroprotective agents or replacing nitrous oxide with other anesthetic compounds that are less neurodegenerative, although more research is needed since these alternatives are still under investigation. Xenon, for example, has been found to deliver neuroprotective effects when taken alongside common anesthetics and is currently undergoing clinical trials in hypoxic neonates. Melatonin is postulated to have supplemental neuroprotective effects when taken in conjunction with classical anesthetics given that it appears to decrease homocysteine production in animal models. Remifentanil may be the anesthetic replacement of choice based on recent but limited studies proving its speed of recovery, analgesic properties, and lack of interference with cerebral blood flow. (Savage & Ma) Safety of nitrous oxide administration is evaluated by examining its minimum alveolar concentration, or MAC, which represents the concentration of gas at 1 atmosphere that leaves 50% of patients unresponsive to a surgical stimulus. This concept is essentially the inhaled anesthetic variant of the classical ED50 measurement. Nitrous oxide’s MAC is 104%. The general safe dosing window for successful anesthesia is between 0.5 to 2.0 MAC units, with a minimum of 1.5 to 2.0 MAC units required to guarantee anesthesia in all patients. (Becker & Rosenberg)

The Hippocratic oath demands that physicians, above all else, do no harm. This undoubtedly makes anesthetic treatment a delicate subject because we must the balance between the benefits of reducing physical harm during treatment and the risk of toxicological harm in the long term. Nitrous oxide has been used for decades as an anesthetic agent and the literature continues to demonstrate that it has neurotoxic effects. Even still, there is no doubt that it is the most widely used agent in anesthetic practice and there is merit to its alternative approaches are bound to arise that will ensure adequate anesthesia without the risk of neurotoxicity.

References

Becker, D. E., & Rosenberg, M. (2008). Nitrous Oxide and the Inhalation Anesthetics. Anesthesia Progress, 55(4), 124–131. http://doi.org/10.2344/0003-3006-55.4.124

Brüne, B. (2003). Nitric oxide: NO apoptosis or turning it ON? Cell Death Differ Cell Death and Differentiation, 10(8), 864-869.

Emmanouil, D. E., & Quock, R. M. (2007). Advances in Understanding the Actions of Nitrous Oxide. Anesthesia Progress, 54(1), 9–18. http://doi.org/10.2344/0003- 3006(2007)54[9:AIUTAO]2.0.CO;2

Rizzi, S., Carter, L. B., Ori, C., & Jevtovic-Todorovic, V. (2008). Clinical Anesthesia Causes Permanent Damage to the Fetal Guinea Pig Brain. Brain Pathology (Zurich, Switzerland), 18(2), 10.1111/j.1750–3639.2007.00116.x. http://doi.org/10.1111/j.1750-3639.2007.00116.x

Rooks, J. P. (2011). Safety and Risks of Nitrous Oxide Labor Analgesia: A Review. Journal of Midwifery & Women’s Health, 56(6), 557-565.

Savage, S., & Ma, D. (2014). The Neurotoxicity of Nitrous Oxide: The Facts and “Putative” Mechanisms. Brain Sciences, 4(1), 73-90.

Schmitt, E. L., & Baum, V. C. (2008). Nitrous oxide in pediatric anesthesia: Friend or foe? Current Opinion in Anesthesiology, 21(3), 356-359.

Zhen, Y., Dong, Y., Wu, X., Xu, Z., Lu, Y., Zhang, Y., . . . Xie, Z. (2009). Nitrous Oxide Plus Isoflurane Induces Apoptosis and Increases β-Amyloid Protein Levels. Anesthesiology, 111(4), 741-752.

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Amphetamine-induced acute neurotoxicity: molecular, morphological, and epidemiological findings. 

The nervous system is a structurally delicate arrangement of neural cells that engage in very precise signaling behavior. It serves as the body’s command center to facilitate an incredible amount of processes via the transmission of nerve impulses. Given its importance and physiological relevance, there is no doubt that systemic disruptions would likely result in alarming health consequences. Toxicology is often directly implicated within nervous system dysfunction and is thusly important to understand for the maintenance of one’s neurological health. Indeed, toxicants can contribute to a host of neurological deficits. One phenomenon commonly discussed in the literature is acute neurotoxicity, which entails the ingestion of a toxic agent that negatively impacts neurotransmission processes. Amphetamines are a perfect example of stimulatory chemicals that affect neurotransmission. The acute neurotoxic effects associated with these chemicals are proven to cause significant neurological problems at the synaptic and morphological level.

Amphetamines have been used by humans for millennia but were identified as excitatory CNS agents in the 1930s. It was around this time that reports of elevated use began to surface. They are often abused to produce feelings of euphoria, increase energy levels, and suppress appetite. They operate on the CNS at the neuronal level by impacting the synaptic transmission of catecholamines. (Ellinwood et. al) A variety of amphetamine drugs exist and specific variants are usually analyzed within the literature to understand the impact on nervous system function at a detailed level. Methamphetamine is an example of a psychoactive drug within this class that can cause acute neurotoxic effects. It impacts the brain’s neurotransmitter systems by elevating the generalized release of dopamine and serotonin as well as the striatal release of glutamate. These neurotoxic effects can ultimately result in long-term damage to axon terminals within deep-brain regions and the prefrontal cortex. (Yu et. al) While dopamine is the most commonly discussed neurotransmitter linked to amphetamine disruption, recent findings have revealed that these drugs can also impact GABA neurotransmission. GABA is the primary inhibitory neurotransmitter within the CNS and is involved in a variety of neuro- homeostatic processes. Amphetamines can affect GABA neurotransmission at the receptor level by influencing the functionality of GABAA receptors (used for rapid transmission of inhibitory effects) via competitive binding and GABAB receptors (used for slow inhibitory post-synaptic currents) by depressing their signaling strength. (Jiao et. al) The impact of amphetamines on serotonin has also been evaluated using animal models. Nonhuman primates administered low doses of MDMA appear to suffer from serotonergic depletion that lasts for several weeks. Additionally, serotonergic axon diameter appears to undergo reduction in multiple brain regions following similar MDMA doses. (Gouzoulis-Mayfrank & Daumann). This finding has remained consistent across a variety of animal models and is of special importance given that axonal diameter is directly linked with neural transmission speed.

Various hypotheses have been generated regarding the mechanistic action of amphetamine neurotoxicity. It is known that amphetamines are transported into the cytoplasm via presynaptic transporters, which then occupy the vesicular monoamine transporter (VMAT2) to increase dopaminergic and serotonergic concentrations. These elevated concentrations effectively reverse the otherwise regular transportation of neurotransmitters to the extracellular space. (Itzhak & Achat-Mendes) Toxicity to dopaminergic and serotonergic terminals can be monitored via neurochemical markers by quantifying the rate-limiting enzymes of both neurotransmitters. Specifically, decreases in tyrosine levels (corresponding to dopamine) and tryptophan hydroxylase levels (corresponding to serotonin) are indicative of damage that can result from the acute neurotoxic effects of methamphetamines. (Yu et. al) In the context of GABA, amphetamines are postulated to disrupt inhibitory activity by impairing the function of pyramidal cells within the prefrontal cortex through apoptotic methods. (Jiao et. al) Amphetamines have also been found to project dopamine from the ventral tegmental area to the prefrontal cortex through the mesolimbic pathway, which is of concern because dopamine receptors located on glutamatergic terminals in this pathway can elevate glutamate release in response to dopamine buildup. (Jiao et. al) It has been proposed on a general level, however, that the broad neurotoxic effects resulting from amphetamine ingestion likely result from interplay of hyperthermia, glutamate release, reactive oxygen and nitrogen species, apoptosis, and dopamine quinone. (Yu et. al)

Since much of the acute neurotoxic effects resulting from amphetamine use are discussed at the cellular and molecular level, a valid question to ask would be how these proposed mechanisms translate to organ-wide dysfunction. While acute neurotoxicity does primarily pertain to the disruption of signal transmission, such effects can lead to significant morphological implications within the CNS. Chromatolysis of the brain stem, cortex, and striatum has been noted in response to consistent MDMA dosing, which is a clear histopathological indicator of brain damage. (Ellinwood et. al) Mephedrone (i.e. ‘bath-salts’), another intense amphetamine variant, is proven to cause reductions in striatal D2 density and damage at dopaminergic and serotonergic nerve endings within the frontal cortex and hippocampus. (Martinez-Clemente et. al) Amphetamine administration also damages dopaminergic nerve endings within the striatum of adult nonhuman primates. (Ricuarte et. al) PET studies have validated the destruction of dopamine transporters within the caudate and putamen following methamphetamine use as well. (Itzhak & Mendes). It is also important to note that the regrowth of serotonergic axons following amphetamine-induced axonal damage has proven incomplete or inadequate in the hippocampus and different cortical areas of some rodent models, suggesting long-term implicational effects. (Gouzoulis-Mayfrank & Daumann) In addition to the destruction of dopaminergic and serotonergic terminal transporters, there is also some evidence that the acute neurotoxic effects of amphetamines may be accompanied by overt cell death, although more research is still needed. This cell death hypothesis has been validated in different populations of GABA interneurons. Amphetamine-induced apoptosis has also been evaluated with respect to its relevance in cell death pathways (such as Fas/FasL) and DNA damage. (Yu et. al)

The neurotoxic effects of amphetamines are dose and time-dependent. Given the illegality and danger of these drugs, dosing information has been analyzed primarily in animal models. Studies have verified that a single 30 mg/kg dose of an amphetamine drug can result in reductions of glutamine, glutamate, and GABA in the corpus striatum as well as circulatory disruptions of down-regulation in glutamine/glutamate and GABA/glutamate ratios. (Jiao et. al) Long-term high dosing of methamphetamine in rats, specifically 40 mg/kg for 7 days, has shown clear signs apoptotic GABA activity within striatal regions. (Jayanthi et. al) No formal studies have been carried out to determine the exact LD50 of amphetamines but it is assumed to be anywhere between 15 to 180 mg/kg. (Amphetamine) Acute neurotoxic effects can be treated with a variety of drugs including minocycline, parkin, endocannabinoids, and more although the therapeutic value of many of these agents is still investigational. Minocycline crosses the blood-brain barrier to produce neuroprotective effects via microglial activation and proliferation. Parkin is used to protect dopaminergic neurons against a variety of cellular insults (including amphetamine toxicity) by shielding neuronal cell bodies. Use of the endocannabinoid system, though still under investigation, may offer neuroprotective properties against amphetamines by inhibiting the reduction of dopamine precursor tyrosine hydroxylase levels. (Yu et. al) The epidemiology of illicit amphetamine use is difficult to determine but it is estimated that 13 millions use amphetamine-type drugs without medical supervision with the most common demographic being single Caucasian males. (Handly)

While amphetamines have been around for millennia, we are only now beginning to understand the neurological concerns that come their use. Based on current findings, it is safe to say that the strict laws embodying amphetamine usage are well merited. Indeed, research on this class of stimulants has been an ongoing endeavor for decades and continued scientific study will reveal great translational insight on human use and abuse.

References

Amphetamine. (n.d.). Retrieved March 07, 2016, from http://toxnet.nlm.nih.gov/cgi- bin/sis/search/a?dbs hsdb:@term @DOCNO 3287

Ellinwood, E. H., King, G., & Lee, T. H. (2000). Chronic Amphetamine Use and Abuse. Retrieved March 07, 2016, from http://www.acnp.org/g4/gn401000166/ch162.htm

Gouzoulis-Mayfrank, E., & Daumann, J. (2009). Neurotoxicity of drugs of abuse – the case of methylenedioxy amphetamines (MDMA, ecstasy), and amphetamines. Dialogues in Clinical Neuroscience, 11(3), 305–317.

Handly, N. (2015, February 2). Amphetamine Toxicity. Retrieved March 07, 2016, from http://emedicine.medscape.com/article/812518-overview#a6

Itzhak, Y., & Achat-Mendes, C. (2004). Methamphetamine and MDMA (Ecstasy) Neurotoxicity: ‘of Mice and Men’ IUBMB Life (International Union of Biochemistry and Molecular Biology: Life), 56(5), 249-255.

Jiao, D., Liu, Y., Li, X., Liu, J., & Zhao, M. (2015). The role of the GABA system in amphetamine-type stimulant use disorders. Frontiers in Cellular Neuroscience Front. Cell. Neurosci., 9.

Jayanthi S., Deng X., Noailles P. A., Ladenheim B., Cadet J. L. (2004). Methamphetamine induces neuronal apoptosis via cross-talks between endoplasmic reticulum and mitochondria-dependent death cascades. FASEB J. 18, 238–251. 10.1096/fj.03-0295com

Martínez-Clemente, J., López-Arnau, R., Abad, S., Pubill, D., Escubedo, E., & Camarasa, J. (2014). Dose and Time-Dependent Selective Neurotoxicity Induced by Mephedrone in Mice. PLoS ONE, 9(6).

Ricaurte, G. A. (2005). Amphetamine Treatment Similar to That Used in the Treatment of Adult Attention-Deficit/Hyperactivity Disorder Damages Dopaminergic Nerve Endings in the Striatum of Adult Nonhuman Primates. Journal of Pharmacology and Experimental Therapeutics, 315(1), 91-98.

Yu, S., Zhu, L., Shen, Q., Bai, X., & Di, X. (2015). Recent Advances in Methamphetamine Neurotoxicity Mechanisms and Its Molecular Pathophysiology. Behavioural Neurology, 2015, 1-11.

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Caffeine-induced arrhythmia: molecular, electrophysiological, and epidemiological findings.

Many psychoactive drugs fall under strict legal regulation due to their detrimental effects on various physiological processes. Caffeine, however, is an interesting anomaly in that it is a psychoactive stimulant devoid of any regulatory oversight. There is no legal limit for how much caffeine a person can purchase or consume. Given that over 90% of the US population acquires it on a daily basis, the stimulatory properties are well known by scientists and consumers alike. Indeed, caffeine can offer short-term boosts in mental alertness due to its well-documented effects on the central nervous system. This alone makes it an invaluable resource for students, workers, and athletes. As is often the case, however, taking something for granted can cause its underlying consequences to be overlooked. One such consequence lies within cardiac physiology. Although research results have been variable, a significant amount of data exists to suggest that caffeine has toxic arrhythmic properties when consumed excessively. Often neglected, this concept is important to recognize for any regular consumer’s health. Analysis of caffeine toxicity at the molecular, organ-wide, and epidemiological level can help explain its potential for cardiac arrhythmia.

The systematic IUPAC name for caffeine is 1,3,7-Trimethylpurine-2,6-dione. This chemical agent and its metabolites are thought to induce arrhythmia at the molecular level. The body can metabolize over 25 different variants of caffeine although the three most commonly studied are paraxanthine, theobromine, and theophylline. This metabolic process is mediated with a demethylizing iesoenzyme known as hepatic cytochrome P- 450 (Echeverri et. al). The metabolites exert their arrhythmic effects on the cardiovascular system through the modification of calcium concentrations. Specifically, they promote elevated intracellular Ca2+ release that disrupts the structure and function a protein channel known as the ryanodine receptor 2 (RyRY2) embedded within the sarcoplasmic reticulum. This receptor is responsible for the important excitation- contraction coupling behavior of cardiomyocytes. Research has revealed that high doses of caffeine can compromise RyRY2 efficacy, which leads to calcium leakages from the SR. (Blayney) These leakages then contribute to a phenomenon known as delayed after- depolarization (DAD) where abnormal and spontaneous impulses are generated after an action potential is nearly or fully repolarized. It is important to note that a well- established link exists between DAD and arrhythmia-induced heart failure within the literature. (Fozzard) In an alternate molecular pathway, caffeine is implicated within the brain’s adenosine system. While adenosine binding normally retards heart activity and reduces blood pressure, the antagonistic binding of caffeine inhibits adenosine’s effects and causes an elevation of nerve impulses, which can increase heart rate to tachycardic levels. (Klatsky)

The aforementioned molecular mechanisms have been verified through broader organ studies although the results are not entirely conclusive. Normally, the electrical signal for heart contraction originates within the SA node. This signal propagates across the atria until it reaches the AV node, at which point it spreads out across the ventricles to complete a contraction cycle. Arrhythmia, however, disrupts the coordination of an otherwise elaborately mediated contraction mechanism. How this relates specifically to caffeine has yet to be investigated fully. However, it has been proven in canine models that lower intravenous caffeine injection can induce vagal stimulation leading to mild arrhythmic symptomology. Higher dosing of caffeine within these same models elicits more complex and severe arrhythmic variants including atrial flutter, atrial fibrillation, and multifocal ventricular premature contraction. (Mehta et. al) The existence of caffeine-induced arrhythmia has also been reinforced using electrocardiogram readings in human subjects. Indeed, there are clear indicators of electrophysiological disruption in response to caffeine load. For example, moderate caffeine consumption has been shown to produce small albeit significant prolongations of QRS complexes. It is postulated that further prolongation of these complexes may be linked to arrhythmic toxicity. (Donnerstein) The electrophysiological effects of caffeine have also been analyzed in normal and cardiac patients. Interestingly, findings have revealed that caffeine appears to shorten the refractory period of the right atrium, AV node, and right ventricle while simultaneously elongating the refractory period of the left ventricle. (Dobmeyer) Although far from complete, research at the organ level has offered some insight into the notion that caffeine affects cardiac electrophysiology.

There are a variety of epidemiological studies that have examined the dose- response relationship of caffeine-induced arrhythmia. Results from this line of study have been very controversial and often conflict with molecular and electrophysiological findings. The general consensus is that 100 mg of caffeine can increase alertness while minimizing risk of adverse effects. Blood pressure begins to increase at around 250 mg and the lethal dose of caffeine is believed to be somewhere around 10 g. Heavy daily caffeine use of 500 to 600 mg is often associated with tachycardic symptoms. (Pelchovitz) The most relevant arrhythmic phenomenon is supraventricular tachycardia. Fast-acting medications typically include adenonsine, calcium channel blockers, and beta-blockers. For treatment of longer-term effects, antidotes include beta-blockers, calcium channel blockers, digoxin, and other antiarrhythmic medicines. It is important to note that some research has also suggested that no relationship or an inverse relationship exists between drinking caffeinated beverages and atrial fibrillation. The figure below published by Pelchovitz et. al (summarizing the findings of multiple epidemiological human studies) proves that the epidemiological link between both variables is lacking:
Screen Shot 2016-02-24 at 7.17.36 PM

Additionally, with respect to caffeine’s inverse relationship to arrhythmia, Klatsky et. al concluded using cox proportional hazard models in 11,679 study participants (198 of whom were hospitalized for arrhythmia) that consuming caffeine can actually counteract arrhythmia in men, women, whites, blacks, and persons older and younger to 60 years of age. (Klatsky) Thus, current epidemiological data suggests that the proposed link between caffeine and arrhythmia is not concrete.

The mixed data arising from research at the molecular, electrophysiological, and epidemiological level can be conflicting but important nonetheless. As research continues and larger sample sizes are employed, the arrhythmic potential of caffeine overdose will be further understood. Based on current findings, it would be wisest to consume caffeine products by following safe dosing windows proposed by medical and research experts until further knowledge establishes a truly conclusive relationship.

References

Blayney, L. M., & Lai, F. A. (2009). Ryanodine receptor-mediated arrhythmias and sudden cardiac death. Pharmacology & Therapeutics, 123(2), 151-177.

Dobmeyer DJ, Stine RA, Leier CV, Greenberg R, Schaal SF. The arrhythmogenic effects of caffeine in human beings. N Engl J Med 1983; 308: 814-6.

Donnerstein RL, Zhu D, Samson R, Bender AM, Goldberg SJ. Acute effects of caffeine ingestion on signal-averaged electrocardiograms. Am Heart J 1998; 136: 643-6.

Echeverri, D., Montes, F. R., Cabrera, M., Galán, A., & Prieto, A. (2010). Caffeine’s Vascular Mechanisms of Action. International Journal of Vascular Medicine, 2010, 1-10.

Fozzard, H. A. (1992). Afterdepolarizations and triggered activity. Cardiac Adaptation in Heart Failure, 105-113.

Klatsky, A. (2011). Coffee, Caffeine, and Risk of Hospitalization for Arrhythmias. Permj The Permanente Journal, 15(3).

Mehta A, Jain AC, Mehta MC, Billie M. Caffeine and cardiac arrhythmias. An experimental study in dogs with review of literature. Acta Cardiol 1997; 52: 273-83.

Pelchovitz, D. J., & Goldberger, J. J. (2011). Caffeine and Cardiac Arrhythmias: A Review of the Evidence. The American Journal of Medicine, 124(4), 284-289.

Supra ventricular Tachycardia – Medications. (n.d.). Retrieved February 24, 2016, from http://www.webmd.com/heart-disease/tc/supraventricular-tachycardia- medications

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The cardiotoxic potential of anthracycline chemotherapeutics. 

Chemotherapy remains the most common method of cancer treatment alongside radiation and surgery. It involves the administration of drugs that are tailored to induce apoptosis in cancerous cells or inhibit further cancer cell mitotic behavior. Anthracyclines are a class of anti-cancer compounds that were discovered in the 1960s to have anti- tumor properties. They are derived from Streptomyces, a filamentous strain of gram- positive bacteria. There are 5 major drugs within this class of chemicals including Doxorubicin, Epirubicin, Idarubicin, and Valrubicin, which are structural analogs of one another varying only slightly in chemical structure. (Anthracyclines) These different agents have been utilized for multiple cancer variants including leukemia, lymphoma, lung cancer, sarcoma, multiple myeloma, hematologic malignancies, and more. Despite the chemotherapeutic significance of these drugs, they are also known to be toxicologically implicated within the cardiac system. Specifically, their toxic properties compromise cardiomyocyte efficacy, which can jeopardize heart muscle structure and function and in some cases lead to heart failure. (Volkova) The basic physiological and biochemical properties of anthracyclines will be examined to develop a greater understanding of their toxicological significance as it pertains to cardiac muscle.

Anthracyclines are derivatives of Streptomyces that operate by targeting DNA. Specifically, these chemicals intercalate themselves within DNA to form complexes that inhibit the regular activity of important enzymes in protein synthesis such DNA polymerase, RNA polymerase, and topoisomerase II. Upon ingestion, the anthracyclines operate through membrane interaction, specifically with negatively charged phospholipids. It is this interactive mechanism that is postulated to induce cardiomyocyte toxicity, which will soon be discussed. (Lavelle) The anthracyclines can also bind to mitochondrial DNA on tumorous cells to disrupt basic cell functionality. (Lah) It is important to note that research findings have revealed anthracycline activity to be largely- concentration dependent. These agents elicit different effects within in vitro assays depending on their associated concentration, which impacts their clinical effectiveness. More structural research is needed to fully understand these concentration-related differences, however. (Szluwaska)

With respect to cardiac toxicity, the mechanistic action of anthracyclines is well recognized. In fact, toxicological findings have led physicians to limit doxorubicin dosing to 400-450 mg/m2 to minimize cardiac risk. This generalized dosing regimen has been reinforced through clinical trial data, which has revealed that nearly 50% of participants dosed with concentrations above 600 mg/m2 suffer from cardiac events and nearly all patients suffer from cardiac toxicity at concentrations above 800 mg/m2. How exactly do these drugs contribute to heart toxicity, however? The anthracyclines induce morphological changes in the cytoplasm and myocyte loss due to their dilating effects on the sarcoplasmic reticulum. Severe cases are localized specifically to the left ventricle and cause cellular remodeling that can lead to heart failure, although findings for the exact biochemical mechanism underlying this process are not yet conclusive. The most commonly proposed explanation is linked to the production of free radicals. Doxorubicin, for example, forms complexes with iron in mitochondrial membranes, which impacts the sensitivity of mitochondrial Ca2+ channels. This sensitivity alteration then elevates mitochondrial ROS production by modifying membrane potentials. (Rahman) The exact mitochondrial ROS formation process is complex but is generally mediated at the enzymatic level. Enzymes such as NADPH oxidase, cytochrome P-450 oxidase, and xanthine oxidase are responsible for transforming the originally quinone-shaped anthracycline drugs into semiquinone structures through electron reduction. In an effort to restore original structure, the semiquinones react with oxygen through redox reactions, forming oxygen free radicals as byproducts. These free radicals, along with others, engage in peroxidation of myocyte membranes, compromising cardiomyocyte structure and function. In fact, histological studies have revealed through the microscopic observation of myocyte cells that lipid ROS peroxidation produces clear signs of interstitial fibrosis, myofibril loss, vacuole degeneration, and chromatin recession. (Angsutararux) Indeed, these indicators clearly underlie the potential for death of cardiac muscle.

The consequences of free radical production are manifested in episodes of acute or chronic cardiotoxicity. Although incidence of the former is lower, biochemical responses generally include arrhythmia, abnormalities in ST and T segments, acute heart failure, and pericarditis-myocarditis syndrome—an inflammatory disease of the myocardium. Given the nature of chemotherapeutic dosing regimens, however, chronic cardiotoxicity is typically more common. Chronic free radical exposure to cardiac muscle often leads to dysfunction of the left ventricle, repolarization abnormalities of QT segments (i.e. QT dispersion), and acute heart failure. Even after the completion of chemotherapy, late-onset symptomology may not present until decades later. (Pfeffer) These numerous toxic factors largely depend on concomitant conditions including hypertension, diabetes mellitus, liver disease, and previous heart diseases. The magnitude of toxicity is also dependent on the method of anthracycline administration. Rapid administration of anthracyclines leads to saturation in the blood, which provides a higher potential for heart damage than gradual ingestion at longer intervals. (Cardiac Toxicity)

A variety of preventative strategies exist to minimize the degradation of cardiac muscle from anthracycline administration. One such strategy is the simultaneous dosing of beta-blockers, which are known for their cardioprotective effects. Specifically, these drugs weaken toxicity through iron chelation such that the anthracyclines cannot react with iron to form ROS species. Dexrazoxane, an EDTA derivative, is postulated to work using the same principle. Another proposed mechanism is the administration of Renin- Angiotensin inhibitors, which are known to reduce the effects of fibrosis and oxidative stress. Aldosterone antagonists have also been shown to attenuate dysfunction of the left ventricle from chronic anthracycline administration through the inhibition of an epidermal growth factor receptor known as EGFR. (Bloom et. al) Administration of compounds with antioxidant properties is likely the most logical treatment option, however. The goal is to minimize the level of free radical destruction by minimizing peroxidation effects. Cardiac antioxidant compounds frequently mentioned in the literature include prubocol and carvedilol. Interestingly, Vitamin E, a common combatant of oxidative stress in the heart, has not shown therapeutic effects in animal assays with left ventricular dysfunction, however. (Volkova)

Ultimately, the decision to treat any variant of cancer through anthracycline chemotherapeutics is at the discretion of the physician. A competent oncologist must evaluate anthracycline administration using basic risk-benefit analysis principles. Consistent communication between the oncologist and cardiologist will increase the level of benefit and safety resulting from treatment. Understanding the mechanisms behind anthracycline cardiotoxicity, monitoring ventricular dysfunction, and understanding associated risk factors all play an important role in the proper administration of anthracyclines to cancer patients.

References

Angsutararux, P., Luanpitpong, S., & Issaragrisil, S. (2015). Chemotherapy-Induced Cardiotoxicity: Overview of the Roles of Oxidative Stress. Oxidative Medicine and Cellular Longevity, 2015, 795602. http://doi.org/10.1155/2015/795602

Anthracyclines. (n.d.). Retrieved February 16, 2016, from http://chemoth.com/types/anthracyclines

Bloom, M. W., Hamo, C. E., Cardinale, D., Ky, B., Nohria, A., Baer, L., . . . Butler, J. (2016). Cancer Therapy–Related Cardiac Dysfunction and Heart Failure. Circulation: Heart Failure Circ Heart Fail, 9(1).

Cardiac Toxicity. (n.d.). Retrieved February 17, 2016, from http://www.texasoncology.com/cancer-treatment/side-effects-of-cancer- treatment/long-term-side-effects/cardiac-toxicity/#a3

Lah, K. (2011, May 9). Anthracyclines – Mechanism of Action. Retrieved February 16, 2016, from http://www.toxipedia.org/display/toxipedia/Anthracyclines – Mechanism of Action

Lavelle F. [Structure and activity of anthracyclines]. Pathol Biol (Paris). 1987 Jan;35(1):11-9. French. PubMed PMID: 3550604.

Pfeffer, B., Tziros C., Katz, R. (2009) Current Concepts of Anthracycline Cardiotoxicity: Pathogenesis, Diagnosis and Prevention. British Journal of Cardiology, 16(2), 85- 89.

Rahman, A. M., Yusuf, S. W., & Ewer, M. S. (2007). Anthracycline-induced cardiotoxicity and the cardiac-sparing effect of liposomal formulation. International Journal of Nanomedicine, 2(4), 567–583.

Szuławska A, Czyz M. [Molecular mechanisms of anthracyclines action]. Postepy Hig Med Dosw (Online). 2006;60:78-100. Review. Polish. PubMed PMID: 16489295.

Volkova, M., & Russell, R. (2012). Anthracycline Cardiotoxicity: Prevalence, Pathogenesis and Treatment. CCR Current Cardiology Reviews, 7(4), 214-220. Retrieved February 16, 2016.

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An evaluation of the proposed MMR-vaccine Autism connection using Bradford Hill criteria.

Bradford Hill developed a set of 7 criteria to evaluate if correlational findings in scientific studies could be viably translated into causational explanations. Dubbed the ‘Hill Criteria’, these 7 points changed the overall dynamic of investigational studies from the 1960’s onward. Hill’s criteria will be used to scrutinize the proposed connection between childhood vaccination and autism. Although regularly dismissed in the literature, this phenomenon remains a concern for several parents around the world as the incidence of autism continues to increase over time.

Hill’s first criterion indicates that the investigator observe the strength of association between variables for the subject in question. In this particular instance, an outdated and retracted study sparked concern for parents worldwide. The first of many issues compromising the study’s legitimacy was the lack of adequate sample size. Per the first criterion, a small association lacks strength and thusly cannot be definitively indicative of a causational effect. Wakefield, the principal investigator of the study, used a sample size of just 12 children to validate his hypothesis that the measles vaccine could be held accountable for autism affliction (Willingham). This sample size, by modern research standards, is mediocre at best and not credible for generating a scientific conclusion. Hill’s second criterion claims that findings must be consistent. To prove consistency, the data must be replicable in other experimental settings. In Wakefield’s favor, although the trial’s results have not been replicated directly, over 20 different peer- reviewed resources exist that can attest to his proposal that the disruption of gut flora from drug administration is evident in several autistic children. These publications come from reputable journals including but not limited to The Journal of Pediatrics, Brain, Behavior, and Immunity, the Journal of Clinical Immunology, and more (Mercola). Thus, whether the nature of Wakefield’s studies entirely fit criterion 2 is speculative.

The third criterion pertains to biological gradient. Per the CDC, low dosing of the MMR vaccine is proven to have no adverse physiological implications beyond swelling at the injection site, rendering Wakefield’s hypothesis as dose-independent (Thimerosal in Vaccines). This in turn voids the notion of biological gradient. Hill’s fourth criterion suggests the investigator evaluate temporality. This concept also goes against the Wakefield study. In order for temporality to be achieved, the causational agent must precede the expected effect. Ironically, the FDA withdrew Thimerosal-based vaccines from the market in 2001 after Wakefield’s publication as a safety precaution. However, the incidences of autism continued to increase even after the recall, indicating that the effect did not occur in sequence with the proposed causational agent (Thimerosal in Vaccines). The fifth criterion requires there be a reasonable level of plausibility in the proposed biological mechanism of the causational relationship. With respect to the Wakefield study, this can go both ways. Wakefield proposed that Thimerosal causes intestinal inflammation that voids the selective permeability of specific encephalopathic peptides, which are then capable of traversing the bloodstream to reach the CNS and cause developmental effects. At the time, this mechanism may have seemed sound given that the vaccine was known to disrupt bacterial flora. However, subsequent studies, namely one carried out recently at the Abrahamson Research center, revealed that the rubella and measles vaccine does not in fact cause any sort of change in intestinal barrier function (Gerber).

The sixth criterion requires coherence with established knowledge. Several studies existed prior to Wakefield’s publication in 1998 to refute his claims, effectively compromising criterion 6. For example, researchers in Finland used registry data from 1982 to 1986 that proved no evident clustering of findings to suggest a connection between vaccinations and autism. Researchers in England participated in an ecological study from 1979 to 1992 to observe the incidence of autism before and after MMR vaccine introduction and noticed no trend-worthy fluctuations in data (Gerber). Indeed, there was sound demographic evidence to refute the fundamental basis of Wakefield’s hypothesis. The seventh and final criterion encompasses specificity. Wakefield successfully narrowed his hypothesis toward a particular demographic using a specific mechanism of action as an explanation. However, to demonstrate legitimate specificity, there must be no alternative mechanisms to explain the proposed causational relationship. Autism found its way into psychiatric literature over 50 years ago. In the decades of time prior to Wakefield’s publication, there were several proposed alternative mechanisms for autism pathology. One of many examples was a study conducted in 1993 by Lotspiech and Cieranello, which linked cerebellar and limbic growth abnormalities to autistic symptomology (Lotspiech). Courchesne suggested in 1997 that autism might arise from neurogenesis malfunction during gestation (Courchesne). It is evident that several resources existed prior to Wakefield’s publication to detract from the specificity of his study.

While the Wakefield study was ultimately unsuccessful, it was an important event in the history of research. Bradford Hill’s seven guidelines are excellent tools to evaluate the legitimacy of a scientific finding. The Hill criteria make it clear that there were many significant flaws in Wakefield’s work, but they also encourage the research to build upon his findings in hopes to understand autism pathology.

References

Courchesne, E. (1997). Brainstem, cerebellar and limbic neuroanatomical abnormalities in autism. Current Opinion in Neurobiology, 7(4), 568.

Gerber, J. S., & Offit, P. A. (2008, October 14). Vaccines and Autism: A Tale of Shifting Hypotheses. Retrieved January 22, 2016, from http://cid.oxfordjournals.org/content/48/4/456.full

Lotspeich, L. J., & Ciaranello, R. D. (1993). The Neurobiology and Genetics of Infantile Autism. International Review of Neurobiology, 87-129.

Mercola, J. (2010, April 10). Why Medical Authorities Went to Such Extremes to Silence Dr. Andrew Wakefield. Retrieved January 22, 2016, from http://articles.mercola.com/sites/articles/archive/2010/04/10/wakefield- interview.aspx

Thimerosal in Vaccines. (2015). Retrieved January 22, 2016, from http://www.cdc.gov/vaccinesafety/concerns/thimerosal/

Willingham, E., & Helft, L. (2014). The Autism-Vaccine Myth. Retrieved January 22, 2016, from http://www.pbs.org/wgbh/nova/body/autism-vaccine-myth.html

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An overview of the role of mitochondria in physiology and toxicology.

The mitochondria are the backbone of cellular biology. These organelles are the driving force that powers our physiology and health. Within the realm of toxicology, they play an important role in toxication and detoxication processes based on their intricate molecular properties. It is important to note that there is still much research required to truly understand the extent with which their role is involved at a toxicological level, but it nonetheless remains active and extensive research endeavor. The role of mitochondria will be discussed at a cellular and molecular, physiological, and toxicological level.

The mitochondrion is arguably the most pivotal organelle of human life. It utilizes 98% of our inhaled oxygen to power the body’s various systems through an ATP- mediated process. This process is the fueling resource for a variety of physiological functions including muscle contraction, homeostasis, neural, and humoral regulation. (Dutchen) ATP generation is a complicated and multi-stepped yet very much important for any competent biological scientist to understand at least at a basic capacity. The process employed for ATP energy generation is known as cellular respiration, which involves the breakdown of sugar in the presence of oxygen. In regular aerobic conditions, it follows a four-step blueprint: (1) glycolysis, (2) the transition reaction, (3) the Krebs cycle, and (4) the electron transport chain. The initial glycolytic phase entails exactly what the name implies—a glucose sugar is lysed (split) in the cytoplasm of the cell (outside of the mitochondria) to form 2 molecules of pyruvic acid, producing 2 ATP energy units. This step is then followed by the transition reaction, where the resultant pyruvic acid is shuttled to the mitochondria. It is in this organelle that the bulk of energy production takes place. Upon transportation, a critical enzyme known Acetyl CoA metabolizes the pyruvate further. This signals the initiation of step 3, the Krebs Cycle, which occurs within the mitochondrial matrix. Here, the aforementioned Acetyl CoA is stripped of oxygen and hydrogen (in sets of two) to extract electrons for further ATP production until all hydrogen is depleted and only carbon dioxide and water remain as waste products. This produces 4 ATP units, which is minimal in comparison to the final step. Nonetheless, it generates a significant quantity of NADH, an important molecule for the final, high-ATP yielding electron transport chain. In this final step, the electrons generated from step 3 traverse through a comprehensive chain-like network of transporters in the membrane of the mitochondrion, generating approximately 32 ATP units per molecule of glucose. (Cellular Respiration) These molecules are the primary energy source for all of the body’s processes. Thus, it should go without saying that mitochondria are crucial organelles that act as the substrate for this comprehensive energy-producing cellular reaction.

On a more practical level, one may ask how these organelles play a direct part in our physiology. Mitochondria are actively involved in the regulation of blood sugar. They use a complex transduction pathway to facilitate insulin secretion by detecting oxygen tension and other factors. (Duchen) They can serve as thermogenic (i.e. body-heating) resources through uncoupling reactions carried out by specific transporter proteins that are genetically modulated. (Rousset) They are also postulated to work as signaling mechanisms for electrolytic regulation using free radical production, which will later be discussed at a toxicological capacity. (Duchen) Mitochondria can also play more specified roles depending on the cells they comprise. Examples include the synthesis, breakdown, and recycling of biochemicals, the synthesis of sex hormones in the reproductive system, the metabolism of neurotransmitters in the neural system, and the detoxification of ammonia within the urea cycle. (Function of Mitochondria) They are also known to play a role in the sensation of oxygen, influencing the nervous system to modify respiratory exertion in response to varying oxygen levels. (Duchen) Indeed, the importance of mitochondria is manifested at all levels of mammalian physiology.

The mitochondria are an excellent example of how cellular biology underlies toxicology. There are many instances where failure of these organelles can result in indirect or direct toxicity. In an indirect example, Casarett and Doull state that 2,4- dinitrophenol can influence a mitochondrion’s biological environment by infiltrating the mitochondrial matrix space and exploiting the regularly established proton gradient, ultimately causing dysfunction that can lead to toxic effects including hyperthermia and seizures. In another example, the neurotoxin successor MPTP is capable of accumulating within the mitochondria of dopaminergic neurons electrophoretically, causing dysfunction and cell death. On the cellular level, mitochondria are actively involved in the formation of free radicals (reactive oxygen species or ‘ROS’), substances formed by accepting or losing an electron. These are implicated in the electron transport chain, where the oxygen free radical can be formed as a byproduct. O2- is postulated to have toxicological significance as it can undergo a series of subsequent reactions to form the toxically reactive hydroxyl and carbonate anion radicals. This process constitutes to one of three levels of systemic mitochondrial toxicity. Additionally, the electron transport chain can also suffer from enzymatic inhibition as a result of toxicant exposure, effectively halting ATP energy generation. The inhibitory substances responsible for this phenomenon are classified as class A, B, C, and D chemicals. Class A chemicals prevent the delivery of hydrogen ions to the ETC, whereas class B chemicals inhibit the physical transportation of electrons across the chain. Class C agents interfere with oxygen’s delivery to cytochrome oxidase, the terminal transporter of the chain. Finally, class D chemicals interfere with ATP synthase, an enzyme that is key in the ETC. (Casarett & Doull’s Toxicology) In the third and final mechanism, a sustained rise of intracellular calcium ions can occur in a phenomenon known as excitotoxicity. The mitochondria contain a transporter known as the MCU, which contributes to this process by rapidly accumulating calcium across the electrochemical gradient. Such excitotoxicity has been implicated in a variety of neurodengerative disorders in the literature. (Nicholls) Mitochondria have also been proven to play a role in the signaling cascade for apoptosis (planned cell death). Using an intrinsic pathway, these organelles produce a variety of death-inducing factors that are released through specialized pores of the organelle that cleave to transcriptional proteins in the nucleus and cytoplasm of targeted cells, resulting in imminent death. (Parsons)

The importance of mitochondria in cellular biology has long been established. The molecular reactions that occur within the mitochondria are clearly at the focal point of our physiology’s functionality. Indeed, it is clear that cellular respiration and toxicity impact our bodies at the system level. As research continues to delve into mitochondria’s role in toxicity, the biological community will continue to understand the correlates of affiliated diseases.

References

Casarett & Doull’s Toxicology: The Basic Science of Poisons, Eighth Edition: The Basic Science of Poisons, Eighth Edition (Kindle Location 3868). McGraw-Hill Education. Kindle Edition.

Cellular Respiration: Or, How one good meal provides energy for the work of 75 trillion cells. (2004, February 16). Retrieved January 29, 2016, from http://www.biology.iupui.edu/biocourses/N100/2k4 ch7respirationnotes.html

Duchen, M. R. (1999). Contributions of mitochondria to animal physiology: From homeostatic sensor to calcium signalling and cell death. The Journal of Physiology, 516(1), 1-17.

Duchen, M. R. (2004). Roles of Mitochondria in Health and Disease. Diabetes, 53(Supplement 1).

Function of Mitochondria. (n.d.). Retrieved January 29, 2016, from http://www.ivyroses.com/Biology/Organelles/Function-of-Mitochondria.php

Nicholls, D., Budd, S., Ward, M., & Castilho, R. (1999). Excitotoxicity and mitochondria. Biochem. Soc. Symp. Biochemical Society Symposium, 66, 55- 67.

Parsons, M., & Green, D. (2010). Mitochondria in cell death. Essays in Biochemistry. Retrieved January 29, 2016.

Rousset, S., Alves-Guerra, M., Mozo, J., Miroux, B., Cassard-Doulcier, A., Bouillaud, F., & Ricquer, D. (2004, February). The Biology of Mitochondrial Uncoupling Proteins. Retrieved January 29, 2016, from http://diabetes.diabetesjournals.org/content/53/suppl_1/S130.full

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An explanation of arsenic poisoning’s relevance to toxicology history.

Poison use is embedded within the roots of toxicological history. Although formal knowledge of toxicology was by no means scientifically comprehensive centuries ago, the fundamental principles of ingestion and toxicity were still understood and deployed voluntarily for purposes of personal gain including advancement, execution, and score settling. Among the various poisonous agents utilized during this period was arsenic, the so-called ‘king of poisons’. It was the covert weapon of choice from the 8th century BC until the mid-19th century AD. (Smith) Given its timely influence as a toxic agent, it is an important historical entity for any competent or aspiring toxicologist to understand in terms of use and mechanistic action.

Considering the abundance of poisons discovered during the course of history, it would be reasonable to ask why arsenic continually remained the poison of choice. Simply put, arsenic cannot be detected using any of our major sensory systems—it has no color, no flavor, and no odor. This made it the perfect weapon of choice for any assailant wishing to inflict harm without the risk of discovery. In fact, these properties made it so widely exploited of a toxicant during Roman times that its notoriety caught the attention of consul Lucius Cornelius Sulla who would soon outlaw its malicious use in his publication, the Lex Cornelia (82 BC). Despite his efforts, the Spanish Borgias continued to exert their influence and expand their power by using the poison to eliminate the elite Roman class. (Gilbert)

While the modes of action and detection may not have been understood until 19th century, those who used the poison were definitely aware of the physiological implications associated with its ingestion. Arsenic has the potential to significantly compromise a variety of organ systems including the skin, nails, hair, nervous system, blood, urine, respiratory system, and more. With respect to the skin, it can cause exfoliative dermatitis (scaling) and in some cases induce skin cancer. Perhaps the most profound implications lie within the nervous system, however. Ingestion of arsenic leads to significant sensory changes and impairments, namely numbness, tingling, headaches, drowsiness, and confusion. It also weakens the physical capacity of small muscles like the hands and feet. Additionally, it can cause variants of anemia and inflammation of the respiratory mucosa. Indeed, all of these handicapping physical and mental effects explain why it was the toxicant of choice for poisoning.

Although once used as a deliberate poison, arsenic still lingers as a toxicant in the food and water supply of several countries, affecting over 200 million people. The mechanisms behind arsenic poisoning are complex and beyond the scope of basic toxicology. However, there are still some simple mechanisms to understand. Arsenic storage is dose-dependent; that is, higher levels are deposited within the kidney, liver, and lungs, whereas lower quantities accumulate within muscles and neuronal tissue. Its toxic mode of action revolves around the inhibition of enzymatic activity responsible for energy pathways, DNA synthesis, and repair. The poison is linked to the generation of ROS, which are known to exert toxic effects on our physiological systems. Arsenic also easily infiltrates the blood-brain barrier, exerting its toxicological effects on neurotransmitter systems to effectively disrupt neural homeostasis. Arsenic concentrations in the urinary tract can also cause renal and nephrotoxicity via oxidative stress and apoptosis. (Kaur). It has also been linked to carcinogenicity, although the respective molecular mechanisms are not yet understood.

By the mid-19th century, chemist James Marsh developed an effective arsenic- screening test through the use of a simple precipitate reaction. This signaled the means to an end for arsenic poisoning as the procedure gained worldwide acclaim. Nevertheless, arsenic will always remain a hallmark toxic xenobiotic in the history of toxicology proving that pharmacological science can be employed not only for treatment, but also malicious intent.

References

Gilbert, S. (2014, May 30). Arsenic Poisoning. Retrieved February 2, 2016, from http://www.toxipedia.org/display/toxipedia/Arsenic Poisoning

Kaur, T., Singh, A., & Goel, R. (2011). Mechanisms pertaining to arsenic toxicity. Toxicology International Toxicol Int, 18(2), 87.

Ngan, V. (n.d.). Chronic Arsenic Poisoning. Retrieved February 02, 2016, from http://www.dermnetnz.org/reactions/arsenic.html

R., Smith. (n.d.). Arsenic: A Murderous History. Retrieved February 02, 2016, from http://www.dartmouth.edu/~toxmetal/arsenic/history.html