Chapter 6
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Inhibition of Chemical Toxicity and Carcinogenesis by Diallyl Sulfide and Diallyl Sulfone J-Y. Hong, M . C. Lin, Zhi Yuan Wang, E-J. Wang, and Chung S. Yang Laboratory for Cancer Research, College of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08855-0789
The effects of diallyl sulfide (DAS) and its metabolite diallyl sulfone ( D A S O 2 ) on the hepatotoxicity induced by acetaminophen (APAP) as well as on lung tumorigenesis induced by the tobaccospecific carcinogen 4-(methylnitrosamino)-1 -(3-pyridyl)-1 -butanone (NNK) were studied. A P A P at a dose of 0.4 g/kg (for rats) or 0.2 g/kg (for mice) caused a severe hepatotoxicity as manifested by the elevation of serum activities of glutamic-pyruvic transaminase and lactate dehydrogenase, and liver centralobular necrosis. A dose- and time-dependent antidotal effect of oral D A S O 2 against A P A P induced hepatotoxicity was demonstrated; D A S was slightly less effective. In the carcinogenesis experiments, 100% of female A / J mice treated with a single dose of N N K (100 mg/kg, i.p.) developed lung tumors with an average tumor multiplicity (tumors/mouse) of 7.2. Administration of DAS (200 mg/kg/ day, p.o.) for 3 days prior to N N K treatment decreased the lung tumor incidence to 38% and tumor multiplicity to 0.6. A single dose of D A S O 2 (100 mg/kg, p.o.) given 2 hr prior to N N K treatment reduced the lung tumor incidence by 50% and tumor multiplicity by 91%. Metabolic activation of N N K was significantly inhibited in the lung and liver microsomes prepared from DAS-treated mice. These results clearly demonstrate that DAS and D A S O 2 are effective agents against APAP-induced hepatotoxicity and NNK-induced lung tumorigenesis, most probably working by inhibition of the metabolic activation of the related toxicant and carcinogen.
Garlic (Allium sativum) has been used widely in culinary practice and as a popular folk medicine for centuries. A major component of fresh garlic is S-allylcysteine sulfoxide (alliin), which is odorless and water soluble. The enzyme alliinase converts alliin to allicin, which is unstable and can be further transformed to other garlic compounds including diallyl sulfide (DAS). DAS can also be formed during cooking or after ingestion of garlic. The estimated amount of DAS derived from 1 g
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of garlic is 30 to 100 μg (7). It has been reported that DAS prevents 1,2-dimethylhydrazine-induced hepatotoxicity in rats (2) and nuclear aberration in mouse colon cells (3) as well as inhibits the carcinogenesis induced by 1,2-dimethylhydrazine, benzo[a]pyrene, and N-nitrosomethylbenzylamine (4,5). The mechanisms involved in the chemoprevention of DAS, however, were not clearly understood. Studies in our laboratory demonstrate that DAS can be metabolized by liver microsomal enzymes to diallyl sulfoxide (DASO) and then to diallyl sulfone ( D A S O 2 ) (6). Our previous work has demonstrated that DAS and its metabolite diallyl sulfone are potent inhibitors of P450 2E1, an enzyme important for the metabolic activation of carbon tetrachloride, N-nitrosodimethylamine (NDMA), and acetaminophen (APAP) (7,8). When a single dose of DAS or D A S O 2 was given to rats, rather selective modulation of hepatic P450 activities was observed. There were no significant changes in the total P450 content and NADPH-P450 reductase activity in the liver microsomes from DAS- or DAS02-treated rats, but the P450 2B1 activity assayed as pentoxyresorufin dealkylation was greatly induced, whereas the P450 2E1 activity assayed as N D M A demethylation was markedly decreased (7). The decrease of P450 2E1 activity by D A S O 2 occurred much more rapidly than by DAS or DASO (7,8). Studies in vitro with rat liver microsomes revealed that DAS and its metabolites DASO and D A S O 2 were all competitive inhibitors of P450 2E1-catalyzed reactions. In addition, D A S O 2 acted as a suicide inhibitor to inactivate P450 2E1 (6). P450 2E1 is a major P450 enzyme constitutively expressed in the liver and is inducible by acetone, ethanol, fasting and diabetes (9-11). Many known P450 2E1 substrates are industrial solvents and environmental chemicals, including some toxic and carcinogenic compounds such as and N D M A (72). P450 2E1catalyzed metabolic activation is required for CCI4 and N D M A to exert their toxicity and carcinogenicity. Consistent with the inhibitory effects of DAS and D A S O 2 on P450 2E1, our previous study demonstrated that these two compounds were effective agents against CCI4- or NDMA-induced hepatotoxicity in rats (8). In the present communication, we describe the protective effects of DAS and D A S O 2 against APAP-induced hepatotoxicity and 4-(methylnitrosamino)-l-(3-pyridyl)1-butanone (NNK)-induced lung tumorigenesis.
CCI4
Protection Against APAP-induced Hepatotoxicity A P A P is the leading analgesic and antipyretic drug used in the United States. Overdose of A P A P is known to cause hepatotoxicity and nephrotoxicity in humans and laboratory animals (13). Over 90% of A P A P is converted to sulfate and glucuronide conjugates which are subsequently excreted in urine. A small portion of A P A P is metabolized by P450 2E1 and 1A2 enzymes to produce /V-acetyl/7-benzoquinone imine (14,15), which is either detoxified by the formation of gluta thione conjugate or arylates the critical cellular proteins to cause toxicity (16, 17). To study the possbile protection against APAP-induced toxicity by D A S O 2 , male Fisher 344 rats (80-90 g) or Swiss Webster mice (8 weeks old) were intragastrically administered A P A P suspended in 0.5% tragacanth at a dosage of 0.4 g/kg (for rats) or 0.2 g/kg (for mice). The animals were fasted for 16 hr prior to the A P A P administration. D A S O 2 in distilled water was given orally at different time points after A P A P dosing. Twenty-four hr after A P A P treatment, the animals were killed and the hepatotoxicity was evaluated by the determination of the serum
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levels of glutamate-pyruvate transaminase (GPT) and lactate dehydrogenase (LDH) activities as well as the extent of liver necrosis. A P A P caused a significant increase in the levels of serum GPT (5-fold) and L D H (7-fold) activities 24 hr after the treatment. Nearly 75% of the liver was necrotic with a typical centralobular localization. D A S O 2 protected the rats against APAP-induced hepatotoxicity in a doseand time-dependent manner. At a dose of 5 mg/kg given 1 hr after APAP, D A S O 2 effectively prevented the elevation of serum L D H and GPT activities as well as the development of severe liver necrosis. When rats were given 50 mg/kg D A S O 2 1 hr after A P A P dosing, a complete protection was observed. The rats had normal serum GPT and L D H levels and the liver morphology was indistinguishable from that of normal rats. When given 3 or 6 hr after APAP, D A S O 2 exhibited partial protection. The dose- and time-dependent effects of D A S O 2 in protection against APAP-induced hepatotoxicity were also demonstrated in mice. When D A S O 2 (25 mg/kg) was given either immediately or 20 min after A P A P dosing, the A P A P caused mouse death, elevation of serum GPT and L D H activities, and liver necrosis were completely prevented. The same dosage of D A S O 2 showed partial protection when given 1 hr after A P A P and was ineffective when given 3 hr after A P A P dosing. In both rats and mice, DAS was slightly less effective than D A S O 2 in protecting against APAP-induced hepatotoxicity under the same dosing conditions. Inhibition of NNK-induced Lung Tumorigenesis by DAS and D A S O 2 (18) In addition to the P450 2El-catalized reactions, our previous work demonstrated that D A S significantly inhibited the metabolism of N N K in rat lung and nasal mucosa (19,20). N N K is a tobacco-specific nitrosamine formed by nitrosation of nicotine during the processing of tobacco and cigarette smoking. It is a potent car cinogen in rodents and a likely causative factor in tobacco-related human cancers. P450-catalyzed metabolic activation (oc-hydroxylation) is required for N N K to exert its carcinogenic effects. The oxidation of the α-methyl group leads to the for mation of formaldehyde and 4-(3-pyridyl)-4-oxo-butyldiazohydroxide, which can alkylate D N A or be converted to 4-hydroxy-l-(3-pyridyl)-l-butanone (keto alco hol). The oxidation of the α-methylene group leads to the formation of 4-oxo-l-(3pyridyl)-l-butanone (keto aldehyde) and the methylating agent methyldiazohydroxide. In mice, the lung is a major target organ in NNK-induced tumorigenesis (21). To determine the chemopreventive effect of DAS on NNK-induced lung tumorigenesis, female A/J mice were fed AIN-76A diet and were treated at 7 weeks of age with DAS in corn oil for 3 days (200 mg/kg body wt/day, p.o.). Two hours after the final treatment, the mice were either given a single dose of N N K (100 mg/kg body wt, i.p.) and kept for 16 weeks thereafter for the determination of the lung tumor production or killed immediately for the preparation of lung and liver microsomes. Treatment of female A / J mice in the control group with a single dose of N N K resulted in 100% of mice bearing lung tumors with an average tumor multiplicity of 7.2 ± 1.1. Administration of DAS prior to N N K treatment signifi cantly decreased the lung tumor incidence to 38% and the tumor multiplicity to 0.6 ± 0.2. No difference was observed in animal body weights among the two groups. D A S O 2 , when given at a single dose (100 mg/kg) 2 hr prior to N N K treatment reduced the lung tumor incidence by 50% and tumor multiplicity by 91%. A lower
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dose of D A S O 2 (20 mg/kg) caused a 38% reduction in tumor multiplicity but had no effect on the lung tumor incidence. To study the mechanism by which DAS inhibited the NNK-induced lung tumorigenesis, the effects of DAS on the metabolic activation of N N K in the lung and liver microsomes were examined. Treatment of the female A/J mice with DAS for 3 days (200 mg/kg/day) caused a significant inhibition of N N K metabolism in the lungs and livers. The rates of the formation of the N N K a-hydroxylation products keto aldehyde and keto alcohol were decreased by 70-80% in the lung and liver microsomes. In studies in vitro, a dose-dependent inhibition by DAS (0.05-2 mM) on the N N K oxidative metabolism was demonstrated in the mouse lung. This suggests that the decreased metabolic activation of N N K in DAS-treated mice could be partially contributed to the direct inhibition by DAS or its metabolites on the NNK-metabolizing enzyme activity. Discussion The present results clearly demonstrate that D A S and its metabolite D A S O 2 effectively protected the experimental animals from the hepatotoxicity induced by APAP, a known substrate of P450 2E1. DAS and D A S O 2 were shown to be potent inhibitors of P450 2E1 in our previous studies. Therefore, the protective effect of DAS and D A S O 2 against the induced hepatotoxicity could be best explained by their ability to inhibit the P450 2E1-catalyzed metabolic activation of A P A P . Although the major P450 enzymes responsible for the metabolic activation of N N K remain to be identified, N N K α-hydroxylation was significantly inhibited in the lung and liver microsomes prepared from the DAS-treated A / J mice. This is consistent with the observed remarkable chemopreventive effect of DAS or D A S O 2 against the NNK-induced lung tumorigenesis. Mechanisms other than inhibition of the metabolic activation have also been proposed to explain the chemoprotective activity of organosulfur compounds. Trapping of the reactive intermediates could occur by chemical conjugation between the electron-rich sulfur atom and the electrophilic species produced during the metabolic activation (22). Induction of phase II detoxification enzymes such as glutathione S-transferase by DAS and other organosulfur compounds have been reported (23). The importance of these mechanisms in protection by D A S or D A S O 2 against chemical toxicity and carcinogenesis remain to be determined. A recent epidemiological study indicated that frequent consumption of garlic and other allium vegetables is associated with a lower incidence of stomach cancer (24). The practical application of DAS and other garlic components in the prevention of human cancers needs further study. Individuals exposed to P450 2E1 inducers such as alcohol and isoniazid have elevated P450 2E1 levels and might be more susceptible to the hepatotoxicity induced by protoxicants which are metabolically activated by P450 2E1. D A S O 2 , which is odorless and relatively nontoxic, may be useful in protecting against the induced hepatotoxicity in those individuals. Acknowledgements The authors would like to thank Dr. Jinmei Pan and Ms. Dorothy Wong for assistance in the manuscript preparation. This work was supported by NIH Grants CA37037, CA46535, ES03938, ES05022, and Grant 88B18 from the American Institute for Cancer Research.
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