Thomas E. Massey

Biochemical and molecular aspects of mammalian susceptibility to aflatoxin B1 carcinogenicity

Abstract Aflatoxin B1 (AFB1) is a fungal toxin that has been implicated as a causative agent in human hepatic and extrahepatic carcinogenesis. In this review, the mechanisms involved in AFB1 toxicity are delineated, in order to describe the features that make a specific cell, tissue, or species susceptible to the mycotoxin. Important considerations include: (i) different mechanisms for bioactivation of AFB1 to its ultimate carcinogenic epoxide metabolite; (ii) the balance between bioactivation to and detoxification of the epoxide; (iii) the interaction of AFB1 epoxide with DNA and the mutational events leading to neoplastic transformation; (iv) the role of cytotoxicity in AFB1 carcinogenesis; (v) the significance of nonepoxide metabolites in toxicity; and (vi) the contribution of mycotoxin-unrelated disease processes. Although considerable controversy remains about the importance of specific events, a great deal has been learned about biochemical and molecular actions of AFB1.

Effects of N-acetylcysteine on metabolism, covalent binding, and toxicity of acetaminophen in isolated mouse hepatocytes☆☆☆

The effects of N-acetylcysteine (NAC) on the toxicity, conjugate formation, and covalent binding of acetaminophen (pHAA) and its presumed toxic metabolite were studied in suspensions of isolated mouse hepatocytes. Preincubation of liver cells with NAC prior to the addition of pHAA resulted in enhanced protection compared to the concurrent addition of pHAA and NAC, thus indicating a time lag between availability of NAC and exertion of a protective effect. Furthermore, a protective concentration of NAC caused a large increase in the proportion of pHAA plus metabolites found as the glutathione (GSH) conjugate and a decrease in covalent binding of radiolabeled pHAA metabolite to proteins. Thus, it appears that NAC protects against pHAA toxicity by increasing the availability of intracellular GSH.

Mechanisms of aflatoxin B1 lung tumorigenesis.

Although aflatoxin B1 (AFB1) is best known as a hepatocarcinogen, the respiratory system can also be a target of this mycotoxin. In isolated lung cells from rabbits and mice, AFB1 is bioactivated by cytochromes P450, primarily in nonciliated bronchiolar epithelial (Clara) cells. However, mutagenesis experiments suggest that the DNA-binding AFB1 epoxide metabolite can leave the cells of origin, and potentially interact with other cell types. Consistent with DNA adduct studies, AFB1-induced AC3F1 mouse lung tumors contain point mutations at guanine residues in K-ras, with the anticipated bias for the A/Jallele. Furthermore, following AFB1 treatment but prior to tumor development, K-ras mutations occur preferentially in mouse Clara cells. However, in contrast to findings with other carcinogens, AFB1-induced mouse lung tumors demonstrate frequent, but heterogeneously distributed, overexpression of p53 protein as well as p53 point mutations, suggesting a carcinogen-specific response. Unlike lung tissue from mice and rabbits, human peripheral lung bioactivates AFB1 primarily by prostaglandin H synthase-and/or lipoxygenasecatalyzed cooxidation, with activity concentrated in macrophages. In addition, although glutathione S-transferase M1-1 has high specific activity for AFB1 epoxide conjugation, lung tissues from GSTM1-null individuals do not demonstrate diminished rates of conjugation, compared to tissues from GSTM1-positive individuals. In summary, AFB1 tumorigenesis in mice demonstrates unique properties, and processes of bioactivation show significant species differences.Although aflatoxin B1 (AFB1) is best known as a hepatocarcinogen, the respiratory system can also be a target of this mycotoxin. In isolated lung cells from rabbits and mice, AFB1 is bioactivated by cytochromes P450, primarily in nonciliated bronchiolar epithelial (Clara) cells. However, mutagenesis experiments suggest that the DNA-binding AFB1 epoxide metabolite can leave the cells of origin, and potentially interact with other cell types. Consistent with DNA adduct studies, AFB1-induced AC3F1 mouse lung tumors contain point mutations at guanine residues in K-ras, with the anticipated bias for the A/J allele. Furthermore, following AFB1 treatment but prior to tumor development, K-ras mutations occur preferentially in mouse Clara cells. However, in contrast to findings with other carcinogens, AFB1-induced mouse lung tumors demonstrate frequent, but heterogeneously distributed, overexpression of p53 protein as well as p53 point mutations, suggesting a carcinogen-specific response. Unlike lung tissue from mice and rabbits, human peripheral lung bioactivates AFB1 primarily by prostaglandin H synthase--and/or lipoxygenase-catalyzed cooxidation, with activity concentrated in macrophages. In addition, although glutathione S-transferase M1-1 has high specific activity for AFB1 epoxide conjugation, lung tissues from GSTM1-null individuals do not demonstrate diminished rates of conjugation, compared to tissues from GSTM1-positive individuals. In summary, AFB1 tumorigenesis in mice demonstrates unique properties, and processes of bioactivation show significant species differences.

Pulmonary fibrosis induced in the hamster by amiodarone and desethylamiodarone

Associated with amiodarone use is pneumonitis which may progress to life-threatening pulmonary fibrosis. Desethylamiodarone, a metabolite, whose role in the etiology of amiodarone-induced pulmonary toxicity has been unclear, also possesses antiarrhythmic activity and could potentially be used as an antiarrhythmic drug itself. We have used a single intratracheal administration of equimolar amounts of amiodarone or desethylamiodarone (1.83 mumol) to male golden Syrian hamsters to investigate the fibrogenicity of desethylamiodarone. Animals were terminated at 1, 7, 14, 21, and 28 days post-treatment, and toxicity was assessed by measurement of lung hydroxyproline content and by histological techniques. Amiodarone and desethylamiodarone significantly increased lung hydroxyproline content over vehicle control animals by 21 days (33 and 58% respectively). While amiodarone-treated lungs had hydroxyproline contents similar to control levels at 28 days, desethylamiodarone-treated lungs remained elevated (44% over control values). Quantitative histologic examination revealed that lungs from desethylamiodarone-treated animals displayed a greater toxic effect, while trichrome staining confirmed the increased deposition of interstitial collagen in these same animals. These results may be due to the higher affinity of the lung for desethylamiodarone and thus a prolonged exposure. The findings indicate that, in the hamster, both compounds are toxic by this route and that desethylamiodarone is not a nontoxic metabolite. Further, use of desethylamiodarone as an antiarrhythmic agent may not be devoid of the adverse effects associated with amiodarone.

Acetaminophen-induced hypothermia, hepatic congestion, and modification by N-acetylcysteine in mice

Abstract Acetaminophen-induced hypothermia and hepatic congestion, their modification by N -acetylcysteine (NAC) and their relationship to hepatotoxicity were studied in Swiss white mice. Acetaminophen (125–750 mg/kg) and NAC (1200 mg/kg) were administered orally and animals killed at various times up to 9 hr. Body temperature declined before overt liver injury and the associated congestion, thereby indicating that hypothermia was centrally mediated. The magnitude of hepatic congestion was sufficient to cause marked liver enlargement. This phenomenon was a possible cause of observed hypovolemia which may in turn have contributed to early mortality. Hypothermia and/or related CNS effects may also have contributed to the early mortality. Coadministration of NAC with acetaminophen prevented the hepatotoxicity, but only partially protected against the hypothermia. When administered 3 hr after acetaminophen, NAC immediately halted the development of congestion and hepatotoxicity, but reversal of the hypothermia was manifested only after several hours. The consequences of congestion and liver enlargement on the expression of biochemical variables such as covalent binding are discussed. Our results indicate that acetaminophen-induced hypothermia and hepatotoxicity develop separately in mice, and that an appreciation of both events is important in understanding the action of antidotal compounds.

Increased acetaminophen-induced hepatotoxicity after chronic ethanol consumption in mice

The effect of chronic ethanol consumption on acetaminophen (200, 400, and 600 mg/kg) toxicity was determined by maintaining mice for 10 days on diets consisting of chow and one of the following drinking solutions: 10% ethanol + 10% sucrose, 8% sucrose, or tap water. Toxicity as manifested by mortality, liver enlargement, and liver congestion was greatest in the ethanol-treated group. We suggest that the greater mortality was a result of the increased liver congestion and consequent hypovolemia. Despite the increased levels of cytochrome(s) P-450, covalent binding of [3H]acetaminophen reactive metabolite(s) to liver protein was not higher in ethanol-treated animals. This can be explained by the higher initial glutathione concentration and/or ability to replenish glutathione in the ethanol-treated group. We suggest that the enhancement of acetaminophen toxicity by ethanol is the result of an effect of ethanol on hepatocyte membranes which renders the cells more susceptible to toxic injury.

Susceptibility to Aflatoxin B1-Induced Carcinogenesis Correlates with Tissue-Specific Differences in DNA Repair Activity in Mouse and in Rat

To investigate the mechanisms responsible for species- and tissue-specific differences in susceptibility to aflatoxin B(1) (AFB(1))-induced carcinogenesis, DNA repair activities of nuclear extracts from whole mouse lung and liver and rat liver were compared, and the ability of in vivo treatment of mice with AFB(1) to alter repair of AFB(1)-DNA damage was determined. Plasmid DNA containing AFB(1)-N(7)-guanine or AFB(1)-formamidopyrimidine adducts were used as substrates for the in vitro determination of DNA repair synthesis activity, detected as incorporation of radiolabeled nucleotides. Liver extracts from CD-1 mice repaired AFB(1)-N(7)-guanine and AFB(1)-formamidopyrimidine adducts 5- and 30-fold more effectively than did mouse lung, and approximately 6- and 4-fold more effectively than did liver extracts from Sprague-Dawley rats. The susceptibility of mouse lung and rat liver to AFB(1)-induced carcinogenesis correlated with lower DNA repair activity of these tissues relative to mouse liver. Lung extracts prepared from mice treated with a single tumorigenic dose of 50 mg/kg AFB(1) i.p. and euthanized 2 hours post-dosing showed minimal incision and repair synthesis activities relative to extracts from vehicle-treated mice. Conversely, repair activity towards AFB(1)-N(7)-guanine damage was approximately 3.5-fold higher in liver of AFB(1)-treated mice relative to control. This is the first study to show that in vivo treatment with AFB(1) can lead to a tissue-specific induction in DNA repair. The results suggest that lower DNA repair activity, sensitivity of mouse lung to inhibition by AFB(1), and selective induction of repair in liver contribute to the susceptibility of mice to AFB(1)-induced lung tumorigenesis relative to hepatocarcinogenesis.

The influence of alcohol consumption, cigarette smoking, and physical activity on leukocyte telomere length

Background: Telomeres protect from DNA degradation and maintain chromosomal stability. Short telomeres have been associated with an increased risk of cancer at several sites. However, there is limited knowledge about the lifestyle determinants of telomere length. We aimed to determine the effect of three factors, known to be important in cancer etiology, on relative leukocyte telomere length (rLTL): alcohol consumption, smoking, and physical activity. Methods: This cross-sectional study included 477 healthy volunteers ages 20 to 50 years who completed a questionnaire and provided a fasting blood sample. Multiplex quantitative real-time PCR (qPCR) was used to measure rLTL. Regression coefficients were calculated using multiple linear regression while controlling for important covariates. Results: There was no association between alcohol consumption and rLTL. Daily smokers and those in the middle and lower tertile of pack-years smoking had shorter rLTL than never daily smokers (P = 0.02). Data were suggestive of a linear trend with total physical activity (P = 0.06). Compared with the lowest quartile, the highest quartile of vigorous physical activity was associated with longer rLTL. A significant linear trend of increasing rLTL with increasing vigorous physical activity was observed (P = 0.02). Conclusions: Cigarette smoking and vigorous physical activity have an impact on telomere length. Smoking was related to shorter telomere length while vigorous physical activity was related to longer telomeres. Impact: The findings from this study suggest that lifestyle may play an important role in telomere dynamics and also suggest that engaging in healthy behaviors may mitigate the effect of harmful behaviors on telomere length. Cancer Epidemiol Biomarkers Prev; 25(2); 374–80. ©2015 AACR.

Amiodarone-induced disruption of hamster lung and liver mitochondrial function : lack of association with thiobarbituric acid-reactive substance production

Amiodarone (AM) is an efficacious antidysrhythmic agent that is limited clinically by numerous adverse effects. Of greatest concern is AM-induced pulmonary toxicity (AIPT) due to the potential for mortality. Mitochondrial alterations and free radicals have been implicated in the etiology of AM-induced toxicities, including AIPT. Isolated hamster lung and liver mitochondria were assessed for AM-induced effects on respiration, membrane potential, and lipid peroxidation. AM (50-400 microM) stimulated state 4 (resting) respiration at complexes I and II of tightly coupled lung mitochondria, with higher concentrations (200 and 400 microM) resulting in a subsequent inhibition. This biphasic effect of AM (200 microM) was also observed with isolated liver mitochondria. Only inhibition of respiration was observed with AM (50-400 microM) in less tightly coupled lung mitochondria. Based on safranine fluorescence, 200 microM AM decreased lung mitochondrial membrane potential (p < 0.05), while a concentration-dependent (50-200 microM) decrease of membrane potential was observed with liver mitochondria exposed to AM (p < 0.05). Formation of thiobarbituric acid-reactive substances (TBARS) was not altered by AM (50-400 microM) in incubations lasting up to 1 h. These results indicate that lipid peroxidation, as indicated by levels of TBARS, does not play a role in AM-induced alterations in mitochondrial respiration and membrane potential.

Effects of dietary vitamin E supplementation on pulmonary morphology and collagen deposition in amiodarone- and vehicle-treated hamsters.

Amiodarone (AM) is a potent antidysrhythmic agent that is limited in clinical use by its adverse effects, including potentially life-threatening AM-induced pulmonary toxicity (AIPT). The present study tested the ability of dietary supplementation with vitamin E (500 IU d,1-alpha-tocopherol acetate/kg chow) to protect against pulmonary damage following intratracheal administration of AM (1.83 micromol) to the male golden Syrian hamster. At 21 days post-dosing, animals treated with AM had increased lung hydroxyproline content and histological disease index values compared to control (P < 0.05), which were indicative of fibrosis. Dietary vitamin E supplementation for 6 weeks resulted in a 234% increase in lung vitamin E content at the time of AM dosing, and maintenance on the diet prevented AM-induced elevation of hydroxyproline content and disease index 21 days post-dosing. Dietary vitamin E supplementation also decreased hydroxyproline content and disease index values in hamsters treated intratracheally with distilled water, the AM vehicle. These results demonstrate a protective role for vitamin E in an in vivo model of AIPT, and suggest that this antioxidant may have non-specific antifibrotic effects in the lung.