Mukul Das

Plant phenols as invitro inhibitors of glutathione S-transferase(s)

Ellagic acid, a commonly occurring plant phenol, was shown to be a potent in vitro inhibitor of GSH-transferase(s) activity. Other plant phenols such as ferrulic acid, caffeic acid and chlorogenic acid also showed a concentration dependent inhibition of GSH-transferase(s) activity. The I50 values of ellagic acid, caffeic acid, chlorogenic acid and ferrulic acid were 8.3 X 10(-5)M, 14.0 X 10(-5)M, 20.0 X 10(-5)M and 22.0 X 10(-5)M respectively, suggesting that ellagic acid is the most potent inhibitor of all the four studied plant phenols. At 55 microM concentration of ellagic acid, a significant inhibition (35-47%) was observed on GSH-transferase activity towards CDNB, p-nitrobenzyl chloride and 1,2-epoxy-3-(p-nitrophenoxy)propane as substrates. Ellagic acid inhibited GSH-transferase(s) activity in a non-competitive manner with respect to CDNB while with respect to GSH it inhibited the enzyme activity in a competitive manner. Other phenolic compounds purpurogallin , quercetin, alizarin and monolactone also showed a concentration dependent inhibition of the enzyme activity with a I50 of 0.8 X 10(-5)M, 1.0 X 10(-5)M, 8.0 X 10(-5)M and 16.0 X 10(-5)M respectively. These inhibitors of GSH-transferase(s) activity should be useful in studying the in vitro enzyme mediated reactions of exogenous and endogenous compounds.

Skin tumor initiating activity of therapeutic crude coal tar as compared to other polycyclic aromatic hydrocarbons in SENCAR mice.

Crude coal tar (CCT), a complex mixture rich in polycyclic aromatic hydrocarbons (PAHs) including carcinogens such as benzo[a]pyrene (BP), is widely used therapeutically in dermatological practice, particularly in combination with ultraviolet radiation in the treatment of chronic dermatoses such as psoriasis. In this study we analyzed the tumor initiating activity of therapeutic CCT preparation (USP) in a two-stage model system (initiation and promotion) in SENCAR mice. The tumorigenicity of CCT was compared with other conventionally studied carcinogenic PAHs; 7,12-dimethylbenz[a]anthracene (DMBA), 3-methylcholanthrene (MCA), benzo[a]pyrene 7,8-diol (BP-7,8-diol) and benzo[a]pyrene (BP). A single topical application of an initiating dose of CCT (20 microliter), DMBA (39 nmol), MCA (746 nmol), BP-7,8-diol (352 nmol) or BP (396 nmol) was followed by twice weekly application of 12-O-tetradecanoylphorbol-13-acetate (TPA) (3.24 nmol). The first tumor appearance in the CCT treated group was at 6 weeks on test as compared with DMBA and MCA (3 weeks) and BP-7,8-diol and BP (4-5 weeks). In a total of 20 animals after 8 weeks on test the DMBA, MCA, BP-7,8-diol, BP and CCT groups of mice showed 457, 176, 106, 76 and 46 tumors, respectively. After 11 weeks 100% of the mice in each experimental group had developed tumors and the number of tumors/mouse was 24.3, 15.0 9.8, 6.6 and 3.3 in the DMBA, MCA, BP-7,8-diol, BP and CCT groups, respectively. These studies provide first evidence that a single topical application of a therapeutic preparation of CCT possesses skin tumor initiating activity.

Topically applied nitropyrenes are potent inducers of cutaneous and hepatic monooxygenases

The inducibility of skin and liver microsomal cytochrome P-450 dependent aryl hydrocarbon hydroxylase and other monooxygenases by a mixture of nitropyrenes was assessed and compared with the parent non-nitrated compound, pyrene. A single topical application of nitropyrenes to neonatal rats resulted in highly significant induction of aryl hydrocarbon hydroxylase, ethoxycoumarin O-de-ethylase, and ethoxyresorufin O-de-ethylase activities in skin and liver after 24 hours. Inducibility of the skin and liver enzymes was 3.9-5.7 fold and 1.8-10.3 fold respectively. On the other hand, aminopyrine N-demethylase, benzphetamine N-demethylase and epoxide hydrolase activities in the liver were unaffected by topically applied nitropyrenes. Furthermore, treatment with nitropyrenes produced a 1 nm shift to the blue region in the wavelength maximum of hepatic microsomal cytochrome P-450. Topically applied pyrene produced only marginal or no effects on cutaneous and hepatic enzyme activities. Our results suggest that nitration of pyrene, a relatively ineffective enzyme inducer, produces nitropyrenes which are potent inducers of hepatic and cutaneous monooxygenases and they resemble 3-methylcholanthrene in this inducing effect.

7-Ethoxyresorufin-O-deethylase activity in human hair roots: A potential marker for toxifying species of cytochrome P-450 isozymes

Assay systems for the evaluation of carcinogen interaction with human tissues are essential for assessing cancer risk. Human hair roots (HHR) are a readily obtainable epithelial tissue source that have been employed for investigating inherited enzyme activities. In this study HHR were found to possess cytochrome P-450-dependent 7-ethoxyresorufin-O-deethylase (ERD) activity which measures cytochrome P-450 isoenzymes that are highly specific (in the order of greater than 95%) markers for the metabolic activation of many environmental carcinogenic substances such as the polycyclic aromatic hydrocarbons (PAHs). Topical application of PAHs (in liquor carbonis detergens) to the scalp of human volunteers was found to enhance the activity of this enzyme in freshly plucked hair roots. Oral and topical administration of ketoconazole to the same subjects resulted in an appreciable (up to 73%) inhibition of detectable enzyme activity. Our data suggest that measurement of ERD in HHR may be a useful marker for the study of toxifying species of cytochrome P-450 isozymes in human populations.

Benzo(a)pyrene diol epoxide-I-DNA adduct formation in the epidermis and lung of SENCAR mice following topical application of crude coal tar

The levels of benzo[a]pyrene diol epoxide-I-deoxyguanosine (BPDE-I-dG) adduct formation in epidermis and lung of SENCAR mice following the topical application of benzo[a]pyrene (BP) alone, crude coal tar (CCT) alone, and the two combined were determined in an enzyme linked immunosorbent (ELISA) assay using monoclonal antibodies. Topical application of two doses of BP (20 micrograms) at 72-h intervals, with sacrifice 24 h later resulted in the formation of 197 fmol and 205 fmol BPDE-I-dG adducts per mg DNA in epidermis and lung, respectively. Topical application of 0.5 ml CCT alone resulted in the formation of 278 fmol and 410 fmol BPDE-I-dG adducts per mg DNA in epidermis and lung, respectively. Simultaneous topical application of 20 micrograms BP and CCT (0.1-0.5 ml) resulted in substantially lower BPDE-I-dG adducts in the epidermis as well as in the lung. Our results suggest that CCT may contain inhibitors of carcinogen-DNA adduct formation and that topical application of CCT produces greater effects on DNA-adduct formation in lung than in epidermis. Thus the cancer-causing potency of the polycyclic aromatic hydrocarbons (PAHs) in CCT may be reduced by other anticarcinogenic constituents present in CCT and systemic absorption of carcinogenic PAHs in CCT applied to skin might have tumorigenic effects in other tissues.

Comparative effects of topically applied nitrated arenes and their nonnitrated parent arenes on cutaneous and hepatic drug and carcinogen metabolism in neonatal rats

The effect of a single topical application of several nitroarenes (1-nitropyrene, nitropyrenes mixture, nitrobenzo(ghi)perylene mixture, 3-nitrofluoranthene, nitrofluoranthene mixture, and nitroperylene mixture) and their corresponding parent arenes to neonatal rats on hepatic and cutaneous drug and carcinogen metabolism was studied. Topical application of each nitroarene (10 mg/kg) resulted in significant induction of aryl hydrocarbon hydroxylase (AHH), 7-ethoxyresorufin O-deethylase (ERD), and 7-ethoxycoumarin O-deethylase (ECD) activities in both skin (1.5- to 14.6-fold) and liver (1.3- to 41.9-fold). The induction of these enzymes by each of the nitroarenes was significant when compared to control or to their corresponding parent arenes. Among the nitroarenes studied, 1-nitropyrene was the least effective in inducing enzyme activities. The inducibility in both skin and liver by different nitroarenes tested was in the following order: nitrofluoranthenes mixture greater than 3-nitrofluoranthene greater than nitroperylenes mixture greater than nitrobenzo(ghi)perylenes mixture greater than nitropyrenes mixture greater than 1-nitropyrene. The nitrofluoranthenes mixture and the nitroperylenes mixture were almost as effective as 3-methylcholanthrene (3-MC). Parent arenes were either ineffective or significantly less effective than nitrated arenes in inducing hepatic and/or cutaneous monooxygenase activities. Hepatic and/or cutaneous benzphetamine N-demethylase (BPD), NADPH cytochrome c reductase, NADH ferricyanide reductase activities, and the levels of cytochrome P-450 and cytochrome b5, remained unchanged following treatment with either topically applied nitroarenes or arenes. However, a shift of approximately 1 nm to the blue region in the absorption maximum of hepatic cytochrome P-450 was observed in animals treated with nitroarenes. This shift was not evident in the case of 1-nitropyrene. Analysis of benzo(a)pyrene metabolites by high-pressure liquid chromatography revealed a significant enhancement in the production of metabolites by nitroarene-treated rat skin and liver microsomes. Our studies suggest that nitroarenes are inducers of hepatic and cutaneous monooxygenases in neonatal rats after topical administration and that they resemble the 3-MC type of inducers in this regard.

Induction of epidermal NAD(P)H:quinone reductase by chemical carcinogens: A possible mechanism for the detoxification

NAD(P)H:quinone reductase, which plays an important role in the detoxification of carcinogenic metabolites as well as oxidative cellular damage, was found to be present in epidermal cytosol where its specific activity far exceeds (140-160%) the corresponding hepatic value. The effect of topical application of crude coal tar, 3-methylcholanthrene and polychlorinated biphenyl Aroclor 1254, on epidermal and hepatic cytosolic NAD(P)H:quinone reductase activities was investigated in neonatal rats, Sencar and athymic nude mice. A single topical application of each agent resulted in significant increases in epidermal (185%-389%) and hepatic (150-255%) enzyme activities. This inducible enzyme may play an important role in the detoxification of reactive quinone species during the course of malignant neoplasia and against oxidative cellular damage in skin.

Carcinogen metabolism in human skin grafted onto athymic nude mice: A model system for the study of human skin carcinogenesis

Human skin grafted onto athymic nude mice maintains its major histological features and may provide a useful system with which to assess the carcinogen interaction with human skin. Significant differences were observed in basal levels of cytochrome P-450 and cytochrome P-448-dependent monooxygenase activities between human grafted and nude mouse epidermis. Topical application of crude coal tar (CCT) to human skin transplanted onto nude mice resulted in 3.9 & 3.5; 3.2 & 2.9 and 1.1 & 1.2 fold increases in mouse and human epidermal aryl hydrocarbon hydroxylase (AHH), ethoxyresorufin deethylase (ERD) and ethoxycoumarin deethylase (ECD) activities, respectively. CCT applied topically to mouse skin resulted in 27.8 & 6.4; 12.8 & 3.3 and 1.7 & 2.6 fold increases in mouse and human epidermal AHH, ERD and ECD activities, respectively. Topical application of coal tar either onto human transplanted skin or to mouse skin also resulted in substantial induction of hepatic and pulmonary AHH and ERD activities. These studies indicate that human skin grafted onto nude mice preserves its metabolic capacity and offers a useful model system with which to assess the effects of polycyclic aromatic hydrocarbons and CCT on cutaneous xenobiotic metabolism in the human population.

Alterations in Benzo(a)Pyrene Metabolism and its DNA Adduct Formation in Skin of Mice Chronically Exposed to Ultraviolet-B Radiation

Cutaneous xenobiotic metabolizing enzymes including aryl hydrocarbon hydroxylase (AHH), 7-ethoxycoumarin 0-deethylase (ECD), epoxide hydrolase (EH) and glutathione S-transferase (GST) activities were examined in SKH hairless mice chronically irradiated with UVB to induce squamous cell carcinoma (SCC). Enzyme activities in irradiated tumor-bearing skin were compared to those present in the skin of non-irradiated control animals as well as in unirradiated non-tumor bearing skin sites of the SCC-bearing mice. The inducibility of skin AHH and ECD in each set of animals was assessed following a single topical application of coal tar (1 ml/100 gm). Enzyme-mediated binding of 3H-benzo(a)pyrene (BP) and its metabolite 7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE-I) to epidermal DNA was also evaluated. Basal AHH and ECD activities in microsomes for UVB-irradiated SCC-bearing dorsal skin were 4.6 and 4.8-fold lower than those in dorsal skin of non-irradiated control animals. Enzyme activities in non-tumor bearing ventral skin from the UVB-irradiated SCC-bearing mice also were 2.2-2.8-fold lower as compared to activities in. the non-irradiated control animals. The reduction in AHH activity paralleled the levels of enzyme-mediated binding of radiolabeled BP metabolites and of BPDE-I to epidermal DNA. GST activity was found to be increased (173% in non-tumor bearing ventral skin of UVB-irradiated mice whereas no difference in activity between SCC-bearing dorsal skin and dorsal skin of sese control animals could be detected. EH activity was unchanged in each group of animals. Treatment with topically applied coal tar resulted in higher inducibility of AHH and ECU in both SCC-bearing (13-fold) as well as in non-tumor skin sites (6-fold) of UVB-irradiated mice than in skin of control animals (3-fold). Coal tar application also increased the covalent binding of 3H-BP and of the metabolite BPDE-I to skin DNA. This was greater in SCC-bearing dorsal skin (119–129%) than in non-irradiated skin of control animals (48–162%). Our studies suggest that the metabolism of BP by cutaneous cytochrome P-450 dependent monooxygenases is impaired in skin of mice irradiated chronically with UVB. These studies also illustrate the complex interrelationship that exist in target tissue simultaneously exposed to chemical and physical oncogens in the environment.