J BIOCHEM MOLECULAR TOXICOLOGYVolume 17, Number 6, 2003

Garlic Attenuates Chrysotile-Mediated PulmonaryToxicity in Rats by Altering the Phase I and Phase IIDrug Metabolizing Enzyme SystemMohamed Ameen, M. Syed Musthapa, Parveen Abidi, Iqbal Ahmad, and Qamar RahmanFibre Toxicology Division, Industrial Toxicology Research Centre, Lucknow 226 001, Uttar Pradesh, India; E-mail: qrahmanitrc@yahoo.co.in

Received 9 July 2003; revised 8 September 2003; accepted 22 September 2003

ABSTRACT: Asbestos and its carcinogenic propertieshave been extensively documented. Asbestos expo-sure induces diverse cellular events associated withlung injury. Previously, we have shown that treat-ment with chrysotile shows significant alteration inphase I and phase II drug metabolizing enzyme sys-tem. In this study we have examined some poten-tial mechanisms by which garlic treatment attenuateschrysotile-mediated pulmonary toxicity in rat. FemaleWistar rats received an intratracheal instillation of 5 mgchrysotile (0.5 mL saline) as well as intragastric garlictreatment (1% body weight (v/w); 6 days per week).Effect of garlic treatment was evaluated after 1, 15,30, 90, and 180 days by assaying aryl hydrocarbonhydroxylase (AHH), glutathione (GSH), glutathioneS-transferase (GST), and production of thiobarbituricacid reactive substances (TBARS) in rat lung micro-some. The results showed that AHH and TBARS for-mation were significantly reduced at day 90 and day180 in chrysotile treated garlic cofed rats; GSH recov-ered 15 days later to the near normal level and GSTelevated significantly after treatment of garlic as com-pared to chrysotile alone treated rat lung microsome.The data obtained shows that inhibition of AHH activ-ity and induction of GST activity could be contribut-ing factor in chrysotile-mediated pulmonary toxicityin garlic cofed rats. However, recovery of GSH andinhibition of TBARS formation by garlic and its con-stituent(s) showed that garlic may give protection byaltering the drug metabolizing enzyme system. C© 2003Wiley Periodicals, Inc. J Biochem Mol Toxicol 17:366–371, 2003; Published online in Wiley InterScience(www.interscience.wiley.com). DOI 10.1002/jbt.10100

KEYWORDS: Chrysotile; Garlic; Chrysotile; Aryl Hy-drocarbon Hydroxylase; Glutathione; Glutathione S-Transferase

Correspondence to: Qamar Rahman.c© 2003 Wiley Periodicals, Inc.

INTRODUCTION

Asbestos is a known carcinogen [1] and its carcino-genic properties have been extensively documented.Clinical and epidemiological studies have establishedthat asbestos fibres are associated with the developmentof pulmonary interstitial fibrosis, lung cancer, and ma-lignant mesothelioma [2]. Asbestos exposure inducesdiverse cellular events related to lung injury [3,4]. Fora decade, several investigators hypothesized that theprocess of disease development due to asbestos ex-posure in humans as well as in animals may be en-hanced due to coexposure to poly aromatic hydrocar-bons (PAHs) [5,6]. Moreover, the continuous derange-ment in the pulmonary drug metabolizing enzymes byasbestos affects the metabolism and clearance of a va-riety of environmental pollutants reaching the lung [7].Earlier studies from our laboratory have suggested thatchrysotile, a carcinogenic variety of asbestos, alters theactivities of phase I and phase II drug metabolizing en-zymes [8]. Thus, continuous impairment in phase I andphase II enzyme system shows that the capability oflung to metabolically dispose of carcinogens may givethem more time to stay in the system and react withmacromolecules, which may increase the probabilityof cancer [9]. Therefore in present study, attempt hasbeen made to investigate the attenuation of chrysotile-mediated toxicity by garlic treatment.

Extensive literature is available regarding medi-cal uses and chemical composition of garlic. A studyfrom our laboratory shows that diallyl sulfide (DAS), aconstituent of garlic, protects mesothelial cells againstasbestos-induced genotoxicity [10]. By supporting theabove fact Guyonnet et al. [11] reported that garlicconstituents protect against chemically-induced tox-icity and carcinogenesis in animal model. Sparninset al. [12] hypothesized that organosulfur compounds(OSCs) have the key potential of chemoprevention,which differ in their structures with respect to number

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of sulfur atom and/or allyl groups. Moreover, garlichas been reported to have antitumorigenic properties[13] and might inhibit a variety of chemically-inducedcancers, including those of breast [14–16], skin [17],and lung [18,19]. Dially sulfide is an effective inhibitorof benzo(a)pyrene (B(a)P)-induced pulmonary neopla-sia in mice [20,21]. The present investigation was con-ducted to evaluate the modulation of drug metaboliz-ing enzymes in lung microsomes in chrysotile treatedgarlic cofed rats. The objective of our study is to evalu-ate attenuation of chrysotile-mediated pulmonary tox-icity by garlic and its constituents as well as the mech-anism of action.

MATERIALS AND METHODS

Chemicals

Benzo(a)pyrene, 3-hydroxy benzo(a)pyrene, and 2-thiobarbituric acid (TBA) were purchased from Sigma(St. Louis, MO). All the other chemicals/reagents wereof analytical grade and purchased from Sisco ResearchLaboratory or Spectrochem (Mumbai, India).

Asbestos Fibres

Indian chrysotile asbestos was obtained from alocal asbestos factory mined at Cuddapah (AndhraPradesh Mining, Hyderabad, India). Particle size be-low 30 �m was prepared according to Zaidi [22].

Garlic Preparation

Garlic extract 10% (w/v) of garlic bulbs (Alliumsativum single clove variety) was prepared from freshlysliced cloves, ground into paste, and dissolved in deion-ized water. The dose (intragastric, i.g.) given to experi-mental rat was 1% body weight (v/w), 6 days per weekthroughout the experimental period.

Treatment of Animals

Female Wistar-rats (150 ± 10 g), were obtainedfrom Industrial Toxicology Research Centre, Animalbreeding colony, Lucknow, India, and were divided intofour groups. One group received a single intratracheal(i.t.) inoculation of 5 mg chrysotile (in 0.5 mL saline),the second group received chrysotile i.t. as well as gar-lic (i.g., 1% body weight (v/w), 6 days per week). Thirdgroup received only garlic (i.g.), whereas the fourthgroup, which was treated as control group, receivedonly saline. The animals were maintained on standardpellet diet supplied by Amrut feeds, Pune, India, and

given tap water ad labitum. They were housed in anair-conditioned room and maintained at 25◦C on a 12 hlight/dark cycle. Twenty-four hours posttreatment ofgarlic, animals from each group were sacrificed 1, 15,30, 90, and 180 days after exposure to chrysotile by de-capitation; their lungs were removed and homogenizedin 0.25 M sucrose. Lung microsomal and cytosolic frac-tions were isolated by the method of Johannesen et al.[23].

Enzyme Assays

Aryl hydrocarbon hydroxylase (AHH) activity wasassayed by the fluorimetric technique [24] using 3-hydroxy benzo(a)pyrene as a standard. Total glu-tathione (GSH) content was estimated by the methodof Sedlack and Lindsay [25]. Glutathione S-transferase(GST) activity was determined by the spectrophotomet-ric procedure [26] using 1-chloro-2,4-dinitrobenzene(CDNB) as a substrate. Lipid peroxidation (LPO) wasevaluated according to the method of Hunter et al. [27].Protein was estimated calorimetrically by the methodof Lowry et al. [28] using bovine serum albumin as astandard.

Statistical Analysis

The student’s t-test (two tailed) was adopted forstatistical analysis and p < 0.05 was considered as sig-nificant.

RESULTS

Rats postfed with garlic when exposed to chrysotileshowed a decrease in the activity of aryl hydrocar-bon hydroxylase at days 30 (p < 0.05), 90 (p < 0.001),and 180 (p < 0.001) as compared to those treated withchrysotile alone. However, AHH activity was signif-icantly elevated as compared to control rat lung mi-crosome at day 90 and 180 as shown in Figure 1. Ratswhich were not fed with garlic when treated withchrysotile showed remarkable elevation of AHH ac-tivity at days 30 (p < 0.05), 90 (p < 0.001), and 180(p < 0.001), however at day one AHH activity was sig-nificantly (p < 0.001) reduced as compared to control.Rats fed with garlic alone showed significant elevationof AHH activity except at day one compared to control.

Rats which were not fed garlic when exposed tochrysotile showed significant depletion in the level oftotal glutathione as compared to control at day 15–180as shown in Figure 2. However, chrysotile exposed gar-lic cofed rats showed significant recovery of GSH levelsat days 30 (p < 0.05), 90 (p < 0.001), and 180 (p < 0.001)

368 AMEEN ET AL. Volume 17, Number 6, 2003

FIGURE 1. Effect of garlic on chrysotile mediated changes in arylhydrocarbon hydroxylase (AHH) in rat lung microsome. Values aremean ±SE (n = 6). a p < 0.05, c p < 0.001 compared to control; ∗ p <

0.05 and ∗∗ p < 0.001 compared to chrysotile alone treated group.

as compared to those treated with chrysotile alone.However, as compared to controls, decline in GSH levelwas significant throughout the postexposure period.Rats fed with garlic alone showed a similar pattern asthe control group; the induction was not significant ascompared to control.

Rats fed with garlic when exposed to chrysotileshowed induction throughout the postexposure period,as compared to those treated with chrysotile alone; theinduction was significant at days 30, 90, and 180, ascompared to controls (Figure 3). Rats not fed with gar-lic when treated with chrysotile group showed a sig-nificant depletion in glutathione S-transferase at 1 (p <

0.001), 15 (p < 0.01), 30 (p < 0.001), 90 (p < 0.001), and180 (p < 0.001) days, as compared to the respective con-trol groups; however, rats fed with garlic alone showed

FIGURE 2. Effect of garlic on chrysotile mediated changes in glu-tathione (GSH) in rat lung microsome. Values are mean ±SE (n = 6),a p < 0.05, b p < 0.01, c p < 0.001 compared to control; ∗ p < 0.05 and∗∗ p < 0.001 compared to chrysotile alone treated group.

FIGURE 3. Effect of garlic on chrysotile mediated changes in glu-tathione S-transferase (GST) in rat lung microsome. Values are mean+SE (n = 6), a p < 0.05, b p < 0.01, c p < 0.001 compared to control;∗ p < 0.05 and ∗∗∗ p < 0.001 compared to chrysotile alone treatedgroup.

a significant (p < 0.001) elevation throughout the pe-riod of experiment as compared to control.

Figures 4 and 5 show a decrease in the production ofTBARS in rat lung microsomes isolated from chrysotileexposed, garlic cofed rats at days 90 (p < 0.05) and180 (p < 0.001), as compared to chrysotile alone ex-posed group. However, TBARS formation was signif-icant in both chrysotile alone and chrysotile exposedgarlic cofed rats as compared to control group throughout the period of study. Furthermore, the maximum in-crease was recorded at day 1 and day 15, then a slightdecrease was observed, which increased again at day180 in chrysotile alone treated group as compared to therespective control. Significant alteration was observed

FIGURE 4. Effect of garlic on chrysotile mediated TBARS formationin rat lung microsome. Values are mean +SE (n = 6), c p < 0.001 com-pared to control; ∗ p < 0.05 and ∗∗ p < 0.001 compared to chrysotilealone treated group.

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FIGURE 5. Effect of garlic on chrysotile mediated TBARS formation(NADPH induced (0.4 mM)) in rat lung microsome. Values are mean+SE (n = 6), a p < 0.05; c p < 0.001 compared to control; ∗ p < 0.05 and∗∗ p < 0.001 compared to chrysotile alone treated group.

in the activity of AHH and GST in lung microsomesisolated from rats fed with garlic alone compared tocontrol; however, modulation of GSH level and TBARSformation was not significant.

DISCUSSION

The lung is the primary site of entry into the bodyof a wide variety of inhaled xenobiotic. It has immensedefense mechanisms against xenobiotic substances en-tering the body; mechanical, cellular, and enzymaticdefense mechanisms act to eliminate hazardous chem-icals. In the enzymatic defense reaction, the xenobi-otic is first functionalized by phase I enzymes, usu-ally by the CYP enzyme system, and then conjugatedto a more soluble and excretable form by phase II en-zymes, such as glutathione S-transferases, sulfotrans-ferases, and N-acetyl-transferases. Sometimes, how-ever, these enzymes transform an otherwise harmlesssubstance into a reactive form, thus the interest inpulmonary monoxygenase enzymes system has beensteadily growing.

Several investigators have shown that garlic oil orallyl sulfides are able to modulate the enzymatic ac-tivities related to xenobiotic metabolisms [29–32]. Gar-lic and its constituent(s) have been shown to inducethe CYP450 superfamily to near maximal levels exceptCYP1A subfamily [33], since assaying aryl hydrocar-bon hydroxylase is an inducible phenotype of CYP1A1.Hong et al. [34] proposed pretreatment of mice withi.g. doses of DASs, as garlic extract attenuated the2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inductionof pulmonary CYP1A1 apoprotein and mRNA levels.

In accordance with our earlier finding AHH got in-duced significantly in rats exposed to chrysotile 30days postexposure. However, the chrysotile treated gar-lic cofed group showed a significant decline in AHHlevel at days 30, 90, and 180, as compared to chrysotilealone exposed group. The induction was significant ascompared to control group. Recent evidence suggeststhat induction of AHH may be disadvantageous. In-duced AHH levels lead to rapid conversion of non-carcinogenic metabolites to active epoxides; the in-termediate epoxides of poly aromatic hydrocarbonsare generally more carcinogenic than the parent com-pound. Inhibition of epoxide formation therefore maybe desirable, because the inhibition of AHH results inless epoxide formation [35,36]. Increased level of AHHactivity in rats treated with chrysotile alone were sup-pressed by garlic supplementation and could be an im-portant contributing factor in chrysotile-mediated pul-monary toxicity.

Similarly, glutathione redox system is an importantdefense system against oxidative damage; it plays a vi-tal role in protecting the cell against the oxidative stressinduced by xenobiotics [37]. The present investigationindicates that chrysotile (alone) exposed (garlic unfed)rats show a significant decline in GSH levels after 15days compared to that in the respective control. Theseresults were further supported by our earlier studyin that asbestos exposure to the experimental animalswere causes of a decline in glutathione reservoir, whicheventually cause free-radical–mediated pulmonary in-jury [38]. Chrysotile exposed garlic cofed rats showedenhanced GSH level 30 days postexposure as com-pared to chrysotile (alone) treated rats. However, GSHlevel showed significant decline as compared to controlthroughout the postexposure period. Srivastava et al.[39] proposed that garlic and its constituents have thecapability to restore the level of GSH. Moreover, glu-tathione S-transferase activities were significantly ele-vated throughout the postexposure period in chrysotileexposed garlic cofed rats as compared to chrysotilealone treated group. Several investigators attributedthat organosulphur compounds could have the abil-ity to increase the levels of GSH and GST activity intissues, whereas GST and glucuronyl transferase playan important role in the detoxification of xenobiotics[34,40]. The enzymatic conversion of active epoxidesto less reactive intermediates is protective and occursby spontaneous conjugation with glutathione or enzy-matic conjugation by glutathione S-transferase. Bensonand coworkers [41] demonstrated that dietary admin-istration of antioxidants increased the activity of phaseII enzymes such as GST activity in extra hepatic tis-sues, such as lung, stomach, small, intestine, and kidney[42]. Brady et al. [43] have shown that diallyl sulfide in-duces the GST-� subfamily, GST-�, as well as quinone

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reductase (QR). There are many reports showing themarked increase in GST activity produced by the di-etary administration of DAS and DADS in mouse andthis may account for the anticarcinogenic properties ofthese molecules by conjugation of the metabolites ofthe carcinogens [18,35,44]. Numerous studies carriedout in the late 1970s established that enzyme systemsinvolved in xenobiotic metabolism were markedly in-fluenced by nutritional changes in the diet, which aresufficient to alter the response of animals and humansto the toxicity of xenobiotics [45–47].

Lipid peroxidation plays an important role in thepathogenesis of a number of diseases/disorders. Therelationship between peroxidative decomposition ofmembrane polyunsaturated fatty acid (PUFA) and CYPis well established. The present investigation showsthat in chrysotile exposed garlic cofed rats TBARS for-mation was significantly reduced at day 90 and 180,but the mechanism of reduction was not yet clear. De-creased lipid peroxidation observed in chrysotile ex-posed garlic cofed rats is attributed to the presence ofsulfur containing active compounds in the form of cys-teine derivatives in garlic. It is known that Allium speciescontain dialkylsulfides, their oxides and thioles, whichcan trap electrons from other systems [48]. Thus, it pre-vents oxygen radical formation to a certain extent andscavenges free radicals [49,50]. However, enhanced en-dogenous antioxidants after garlic treatment might bea reason for decreased level of TBARS in this study.It regulates the TBARS production levels by destroy-ing 4-hydroxynonenal, a key reactive aldehyde pro-duced during peroxidation [51,52]. Even though themechanism by which garlic attenuates chrysotile me-diated pulmonary toxicity is not clearly known, thereis sufficient evidence to suggest that their effect may, atleast in part, be due to their ability to increase detoxify-ing enzymes and diminish the ROS mediated oxidativestress.

In conclusion, these results suggest that alterationsmediated by garlic in the metabolic disposition ca-pability of lung by modulating phase I and phaseII metabolizing enzymes and reduced lipid peroxida-tion shows garlic may play a vital role in the attenu-ation of fiber-mediated carcinogenic response. Furtherstudies are suggested in this direction to support thishypothesis.

ACKNOWLEDGMENTS

The authors are thankful to Dr. P. K. Seth, Directorfor his keen interest and necessary support. Thanks arealso due to Mr. Mohd. Ashquin, Technical Officer, forhis skillful technical assistance.

REFERENCES

1. Mossman BT, Gee JB. Asbestos related diseases. N Engl JMed 1989;320:1721–1730.

2. Mossman BT, Bignon J, Corn M, Eaton A, Gee J. Asbestos:Scientific development and implication for public policy.Science 1990;247:294–301.

3. Jaurand MC. Mechanism of fibre induced genotoxicity.Environ Health Perspect 1997;105:1073–1084.

4. Manning CB, Vallyathan V, Mossman BT. Disease causedby asbestos: Mechanisms of injury and diseases develop-ment. Int Immunopharmacol 2002;21:191–200.

5. Arif JM, Khan SG, Aslam M, Mahmood M, Rahman Q.Diminution in kerosene mediated induction of drug me-tabolizing enzymes by asbestos in rat lungs. PharmacolToxicol 1992;71:37–40.

6. Kamp DW, Greenberger MJ, Sbalchierro JS, PreusenSE, Weitzman SA. Cigarette smoke augments asbestos-induced alveolar epithelial cell injury: Role of free radi-cals. Free Radical Biol Med 1998;25:728–739.

7. Arif JM, Khan SG, Mahmood M, Aslam M, Rahman Q.Effect of coexposure to asbestos and kerosene soot onpulmonary drug metabolizing enzyme system. EnvironHealth Perspect 1994;102 :181–183.

8. Rahman Q, Khan SG, Ali S. Effect of chrysotile asbestoson cytochrome P450 dependent monoxygenase and glu-tathione S-transferase activities in rat lung. Chem-BiolInteractions 1990;75:305–314.

9. Hinchin RF, Boyd MR. Localization of metabolic activa-tion and deactivation systems in the lung: Significance tothe pulmonary toxicity of xenobiotics. Annu Rev Phar-macol Toxicol 1983;23:217–238.

10. Lohani M, Yadav S, Schiffmann D, Rahman Q. Diallyl-sulfide attenuates asbestos-induced genotoxicity. ToxicolLett 2003;143:45–50.

11. Guyonnet D, Belloir C, Suschetet M, Siess MH, LeBonAM. Liver subcellular fractions from rats treated byorganosulfur compounds from Allium modulate muta-gen activation. Mutat Res 2000;466:17–26.

12. Sparnins VL, Barany G, Wattenberg LW. Effect oforgano sulfur compounds from garlic and onionson benzo(a)pyrene induced neoplasia and glutathioneS-transferase activity in the mouse. Carcinogenesis1988;9:131–134.

13. Milner JA. Garlic: Its anticarcinogenic and antitumori-genic properties. Nutr Rev 1996;54:S82–S86.

14. Amagase H, Schaffer EH, Milner JA. Dietary compo-nents modify the ability of garlic to suppress 7,12-dimethylbenzo(a)anthracene-induced mammary DNAadducts. J Nutr 1996;126:817–824.

15. Schaffer EM, Lin JZ, Milner JA. Garlic powder and allylsulfur compounds enhances the ability of dietary selin-ite to inhibit 7,12-dimethylbenzo (a)anthracene-inducedmammary DNA adducts. Nutr Cancer 1997;27:162–168.

16. Wargovich MJ. Diallyl sulfide, a flavor compounds of gar-lic (Allium sativum), inhibits dimethylhydrazine inducedcolon tumors. Carcinogenesis 1987;8:487–489.

17. Perchellet JP, Perchellet EM, Belman S. Inhibition ofDMBA-induced mouse skin tumorigenesis by garlic oiland inhibition of two tumor-promotion stages by garlicand onion oils. Nutr Cancer 1990;14:183–193.

18. Hu Xun, Singh SV. Glutathione S-transferases of femaleA/J mouse lung and their induction by anticarcinogenicorgano sulfides from garlic. Arch Biochem and Biophy1997;340:279–286.

Volume 17, Number 6, 2003 GARLIC ATTENUATES CHRYSOTILE-MEDIATED TOXICITY 371

19. Fukushima S, Takada N, Hori T, Wanibuchi H. Cancerprevention by organosulfur compounds from garlic andonion. J Cell Biochem 1997;27:100–105.

20. Wattenberg LW. Inhibition of neoplasia by minor dietaryconstituents. Cancer Res 1983;43:2448S–2453S.

21. Wattenberg LW, Sparnins VL, Barany G. Inhibition of N-nitrosodiethylamine carcinogenesis in mice by naturallyoccurring organosulfur compounds and monoterpenes.Can Res 1989;49:2689–2692

22. Zaidi SH. Experimental Pneumoconiosis. Baltimore, MD:Johns Hopkins Press; 1969. pp 94–109.

23. Johannesen K, Depierre JW, Bergstrand A, Dallner G,Ernslter L. Preparation and characterization of totaland smooth microsomes from the lung of control andmethylchloanthrene treated rats. Biochem Biophys Acta1977;496:115–135.

24. Dehnen W, Tomingas R, Roos J. A modified method forthe assay of benzo(a)pyrene hydroxylase activity. AnalBiochem 1973;97:373–383.

25. Sedlack V, Lindsay RH. Estimation of total protein-boundand non-protein sulfhydryl groups in tissue with Ell-man’s reagent. Anal Biochem 1968;25:192–205.

26. Habig WH, Pabst MJ, Glutathione S-transferase. The firstenzymatic step in the mercapturic acid formation. J BiolChem 1974;249:7130–7139.

27. Hunter FE, Gebiciki JM, Hoffsten PE, Weinstein J, ScottA. Swelling and lysis of rat liver mitochondria inducedby ferrous ions. J Biol Chem 1963;238:828–835.

28. Lowry OH, Rosebrough N, Farr AL, Randall RJ. Pro-tein measurement with Folin phenol reagent. J Biol Chem1951;193:265–275.

29. Haber D, Siess MH, Waziers E, Beaune P, Suschelet M.Modification of hepatic drug metabolizing enzymes inrat fed naturally occurring allylsulphides. Xenobiotica1994;24:169–182.

30. Agarwal KC. Therapeutic actions of garlic constituents.Med Res Rev 1996;16:111–124.

31. Wang BH, Zuzel KA, Rahman K, Billington D. Pro-tective effect of aged garlic extract against bromoben-zene toxicity to precision cut rat liver slices. Toxicology1998;126:213–222.

32. Guyonnet D, Belloir C, Suschelet M, Siess MH, LeBonAM. Antimutagenic activity of organosulfur compoundsfrom allium is associated with phase II enzyme induction.Mutat Res 2001;495:135–145.

33. Dragner KH, Nims RW, Lubets RA. The chemopreventiveagent diallylsulfide: A structurally atypical phenobarbi-tal type inducer. Biochem Pharmacol 1995;50:2099–2104.

34. Hong JY, Wang ZY, Smith TJ, Zhou S, Shi S, Pau J, YangCS. Inhibitory effect of diallyl sulfide on the metabolismand tumorigenicity of the tobacco-specific carcinogen 4-(methyl nitrosamine)-1-(3-pyridyl)-1-butanol (NNK) inA/J mouse lung. Carcinogenesis 1992;13:901–904.

35. Wattenberg LW, Leong JL. Inhibition of carcinogenicaction of benzo(a)pyrene by flavones. Cancer Res1970;30:19–22.

36. Wheatley DN. Enhancement and inhibition of the induc-tion by 7, 12-dimethylbenz (a)anthracene of mammarygland tumor in female Sprague-Dawley rats. Br J Cancer1968;22:787.

37. Rahman Q, Abidi P, Afaq F, Schiffmann D, Mossman BT,Kamp DW, Athar M. Glutathione redox system in oxida-tive lung injury. Critical Rev Toxicol 1999;29:543–568.

38. Abidi P, Afaq F, Arif JM, Lohani M, Rahman Q. Chrysotilemediated imbalance in the glutathione redox systemin the development of pulmonary injury. Toxicol Lett1999;106:31–39.

39. Srivastava SK, Hu X, Xia H, Zaren HA, Chatterjee ML,Agarwal R, Sing SV. Mechanism of different efficacy ofgarlic organosulfides in preventing benzo(a)pyrene in-duced cancer in mice. Cancer Letter 1997;118:61–67.

40. Ander MW, Lash L, Dekout W, Flafarra AA, Dohn DR.Biosynthesis and biotransformation of glutathione con-jugates to toxic metabolites. Crit Rev Toxicol 1988;18:311–341.

41. Benson AM, Cha YN, Bueding E, Heine HS, TalalayP. Elevation of extrahepatic glutathione S-transferasesand epoxide hydrolase activities by 2(3)-tes-butyl-4-hydroxyanisole. Cancer Res 1979;39:2971–2977.

42. Kensler TW. Chemoprevension by inducers of carcino-gen detoxification enzymes. Environ Health Perspect1997;105:965–970.

43. Brady JF, Ishizaki H, Fukuto JM, Lim MC, Fadel A, GapacJM, Yang CS. Inhibition of cytochrome P4502E1 by diallylsulfide and its metabolites. Chem Res Toxicol 199l;4:642–647.

44. Jin F, Baillie TA. Metabolism of chemopreventive agentsdiallyl sulfide to glutathione conjugates in rats. Chem ResToxicol 1997;10:318–327.

45. Parke DV. Nutritional requirement for detoxication of en-vironmental chemicals. Food Addit Contam 1991;8:381–396.

46. Choudhury A, Das T, Sharma A. Use of crude extractof garlic (Allium sativum) in reducing cytotoxic effectsof arsenic in mouse bone marrow. Phytotherapy Res1993;7:163–166.

47. Ioannides C. Effect of diet and nutrition on the expressionof cytochrome P450. Xenobiotica 1999;29:109–154.

48. Klanns-Dieter A. Sulfur contents free radicals. In:Nugaard OF Simic, MG, editors. Radio Protectors andAnti-Carcinogens. New York: Academic Press; 1983;pp 23–42.

49. Helen A, Krishnakumar K, Vijayammal PL, AugusthiKT. Antioxidant effect of onion oil (Allium spec.) on thedamages induced by nicotine in rats as compared to �-tocopherol. Toxicol Lett 2000;116:61–68.

50. Dwivedi C, John LM, Schmit DS, Engineer FN. Effectsof oil soluble organosulfur compounds from garlic ondoxorubin-induced lipid peroxidation. Anticancer Drugs1998;9:291–294.

51. Fanelli SL, Castro GD, De Toranzo EG, Castro JA. Mech-anisms of the preventive properties of some garlic com-ponents in the carbon tetrachloride promoted oxidativestress. Diallyl sulfide, diallyl disulfide, allyl mercaptanand allyl methyl sulfide. Res Commun Mol Pathol Phar-mocol 1998;13:163–174

52. Chen L, Hong JY, So E, Hussin AH, Cheng WF, Yang CS.Decrease of hepatic catalase level by treatment with di-allyl sulfide and garlic homogenates in rats and mice. JBiochem Mol Toxicol 1999;13:127–134.