Differences in benzo(a)pyrene metabolism between rodent liver microsomes and embryonic cells.

Differences in benzo(a)pyrene metabolite pattern have been shown by rodent liver microsomes (Sprague-Dawley) and rodent embryo cells from Syrian hamsters and NIH Swiss mice. Rodent liver induced by methylcholanthrene shows marked quantitative variation between species. Additional pattern changes were found in mouse and hamster embryo secondary cultures with a reduction of the K-region metabolites and a marked increase in 9-hydroxybenzo(a)-pyrene. These results are indicative of a region-specific attack on the carcinogen by the cell monooxygenases which is distinct from the liver attack of microsomal enzymes on benzo(a)pyrene. These results suggest that activation and detoxification of benzo(a)pyrene may be species and tissue variable, and susceptibility and resistence to malignant transformation may be predicted on induction of a fortuitous combination of intermediate metabolic steps.

Aryl hydrocarbon (benzo[a]pyrene) hydroxylase: a mixed-function oxygenase in mouse skin.

Mouse skin contains aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity which is inducible by aromatic polycyclic hydrocarbons and benzoflavones. The duration and magnitude of induction, but not the initial kinetics, are dependent on the inducer dose. The cutaneous hydroxylase activity is inhibited by carbon monoxide and requires the presence of NADPH, indicating that the enzyme is one of the mixed-function oxygenases. The highest enzyme activity was found in the superficial layer of skin which contains the sebaceous glands and the upper pilary canals. Enzyme activities were intermediate in the epidermis and lowest in the deeper dermal layers.

Piperine, a major ingredient of black and long peppers, protects against AFB1-induced cytotoxicity and micronuclei formation in H4IIEC3 rat hepatoma cells.

We studied the effect of piperine on the cytotoxicity and genotoxicity of aflatoxin B1 (AFB1) in rat hepatoma cells H4IIEC3/G-(H4IIE) using cellular growth and formation of micronuclei as endpoints. Piperine was earlier shown to inhibit cytochrome P-450-dependent aryl hydrocarbon hydroxylase and 7-methoxycoumarin demethylase activities in preparations of these cells with 1/2 maximum inhibition at 30-50 microM (Singh J. and Reen R.K., Current Science, 66, 365-369, 1994). The results of the present study showed that AFB1 inhibited the growth of H4IIE cells with an ED50 of 15 nM. Piperine markedly reduced the toxicity of the mycotoxin. Thus at 100 microM piperine largely restored the rate of growth of the cells. Likewise, piperine reduced the AFB1-induced formation of micronuclei in a concentration-dependent manner. Piperine itself was not toxic to the cells up to a concentration of almost 100 microM. The results suggest, that piperine is capable of counteracting AFB1 toxicity by suppressing cytochromes P-450 mediated bioactivation of the mycotoxin.

Benzo[a]pyrene initiates enzyme-altered islands in the liver of adult rats following single pretreatment and promotion with polychlorinated biphenyls.

Treatment of adult female rats with a single dose of benzo[a]pyrene (BaP) fails to initiate preneoplastic enzyme-altered islands in their livers. Treatment with polychlorinated biphenyls (PCBs) at a dose which strongly induces aryl hydrocarbon(BaP)hydroxylase prior to BaP application and followed by promotion with PCBs causes the appearance of about 9 adenosine-5'-triphosphatase-deficient and 7 gamma-glutamyltranspeptidase-positive islands/cm2 after 12 weeks. PCB-pretreatment or promotion alone did not increase the BaP-dependent formation of islands above that of the PCB-treated controls (2-3 islands/cm2). The results suggest that upon alteration of its metabolism BaP causes the formation of preneoplastic lesions in the liver which become manifest by promotion.

Metabolism of fluperlapine by cytochrome P450-dependent and flavin-dependent monooxygenases in continuous cultures of rat and human cells.

The metabolism of fluperlapine, a neuroleptic dibenzazepine derivative with a N-methyl-piperazinyl substituent, was investigated in continuous cultures of rat and human cells which express various cytochrome P450-dependent monooxygenase activities. The differentiated rat hepatoma cells H4IIEC3/G- and their variants 2sFou and FGC-5 metabolized fluperlapine predominantly by N-oxygenation and only to a minor degree by N-demethylation or glucuronidation of primary phenolic products. Total fluperlapine metabolism in dedifferentiated rat hepatoma cells H5 and partially differentiated human hepatoma cells HepG2 was much smaller than in the differentiated rat hepatoma lines. This was primarily attributable to their low capacity for N-oxygenation. Human lung adenocarcinoma lines NCI-H322 and NCI-H358 formed only trace amounts of fluperlapine N-oxide. Pretreatment of 2sFou cells with benz(a)anthracene, phenobarbital or dexamethasone markedly increased the formation of N-demethylated and glucuronidated products but did not affect the rate of N-oxide formation. Guanethidine and cysteamine, inhibitors of flavin-dependent monooxygenase activity, reduced fluperlapine N-oxidation more strongly than aldrin epoxidation, a marker for cytochrome P450 activity. In contrast, n-octylamine inhibited aldrin epoxidation but was without effect on fluperlapine N-oxygenation. The results suggest that certain cells in continuous culture are capable of expressing flavin-dependent monooxygenase(s) in addition to cytochrome P450-containing monooxygenases. Such cells may offer useful systems for studying the oxidative metabolism of a broad spectrum of xenobiotics and analysing the importance of the two oxygenation reactions for the biological effects of their substrates.

Expression of glutathione S-transferase and phenol sulfotransferase, but not of UDP-glucuronosyltransferase, in the human lung tumor cell lines NCI-H322 and NCI-H358.

The expression of xenobiotic-metabolizing enzymes was studied in the human lung tumour cell lines NCI-H322 and NCI-H358. These cells are derived from adenocarcinomas and exhibit features of Clara cells and alveolar type II cells, respectively. Examination of the in vitro activities showed that both cell lines lack UDP-glucuronosyltransferase against the substrates 3-hydroxybenzo[a]pyrene (3-OH-BaP) and 4-hydroxybiphenyl (4-OH-Bph) and that in vitro conjugation of sulfate with 3-OH-BaP was only just detectable. In contrast, both cell lines showed fairly high levels of glutathione-S transferase activity with the substrate 1-chloro-2,4-dinitro-benzene (54.4 and 83.0 nmol/min X mg protein, respectively) and of glutathione (81 and 41 nmole/mg protein, respectively). The metabolic capacity of intact NCI-H322 and NCI-H358 cells was examined using benzo[a]pyrene (BaP) and 3-OH-BaP as substrates. The cell lines formed sulfate conjugates from 3-OH-BaP (4.5 and 0.4 pmol/min X mg protein, respectively) but did not produce any detectable glucuronides. When cultures of the two cell lines were exposed to BaP, phenolic products accumulated in the growth medium. NCI-H322 cells also formed some sulfate conjugates, whereas such conjugates were barely detectable in the medium of NCI-H358 cells. In contrast A549, a human pulmonary adenocarcinoma cell line known to contain UDP-glucuronosyltransferase activity, efficiently conjugated 3-OH-BaP to glucuronic acid and converted the primary phenolic products formed from BaP to glucuronides. Thus the NCI-H322 and NCI-H358 cells are exceptional in that they possess no or very little glucuronosyltransferase activity but exhibit appreciable monooxygenase activity. The cell lines may therefore be of interest for examining the biological effects of potentially toxic chemicals which are otherwise detoxified by glucuronic acid conjugation. The cells may also be useful as test systems for evaluating the potential cytotoxicity and genotoxicity of chemicals to human lung.

Impairment of UDP-glucose dehydrogenase and glucuronidation activities in liver and small intestine of rat and guinea pig in vitro by piperine.

The effects of piperine, a major ingredient of black pepper, on UDP-glucose dehydrogenase (UDP-GDH) and glucuronidation potentials of rat and guinea pig liver and intestine were studied. Piperine caused a concentration-related strong inhibition of UDP-GDH (50% at 10 microM) reversibly and equipotently, in both tissues. Partially purified rat liver UDP-GDH was used to obtain the kinetic values at pH optima of 9.4 and 8.6. At pH 9.4: KmUDP-glucose = 15 microM, Vmax = 5.2 nmol NADH/min/mg protein, Ki = 6 microM. With NAD, a Ki of 16 microM was obtained. At pH 8.6: Km = 35 microM, Vmax = 7.5 nmol, Ki = 15 microM. In all of these cases, piperine caused non-competitive inhibition. Data from structure-activity comparisons of piperine analogs indicated that the presence of conjugated double bonds in the side chain of the molecule is a factor in piperine inhibition. However, the UDP-glucuronic acid (UDPGA) contents were decreased less effectively by piperine in isolated rat hepatocytes compared with enterocytes of guinea pig small intestine. Piperine at 50 microM caused a marginal decrease of UDPGA in hepatocytes when the rate of glucuronidation of 3-hydroxybenzo[a]pyrene (3-OH-BP) decreased by about 40%. The decrease obtained at 10 microM piperine in intestinal cells was comparable to that obtained at 50-100 microM in hepatocytes. UDP-glucuronosyltransferase (UGT) activities towards 3-OH-BP (UGT1A1) and 4-OH-biphenyl (UGT2B1) were also determined. Piperine did not affect the rate of glucuronidation of 4-OH-biphenyl in rat liver, whereas that of 3-OH-BP was impaired significantly. In guinea pig small intestine, both these activities were inhibited significantly requiring less than 25 microM piperine to produce a more than 50% inhibition of UGT(s). The results suggested that (i) piperine is a potent inhibitor of UDP-GDH, (ii) inhibition is offered exclusively by the conjugated double bonds of the molecule, and (iii) piperine exerts stronger effects on intestinal glucuronidation than in rat liver.

Arylamines suppress their own activation and that of nitroarenes in V79 Chinese hamster cells by competing for acetyltransferases.

The effect of 2-aminofluorene (2-AF) on the toxicity of 2-aminoanthracene (2-AA) and 1,6-dinitropyrene (1,6-DNP) was studied in N-acetyltransferase-proficient V79-NHr1A2 cells genetically engineered for the expression of cytochrome P4501A2, and in wild-type V79-NH cells. 2-AA inhibited the growth of V79-NHr1A2 cells and induced the formation of micronuclei at concentrations of 0.1 to 1.0 microM, but was virtually without toxic effects at a concentration of 10 microM. Addition of 2-AF protected against the cytotoxic and genotoxic effects elicited by low concentrations of 2-AA. Half-maximum protection was observed at 0.2 to 0.5 microM 2-AF. The arylamine also prevented the cytotoxicity caused by 1,6-DNP in V79-NH cells and completely suppressed the formation of 1-acetylamino-6-nitropyrene from 1,6-DNP in these cells. The results indicate that arylamines and related N-hydroxyarylamines are substrates for the same acetyltransferase in V79-NH cells. In consequence, arylamines are capable of suppressing the activation of their proximate cytotoxic and genotoxic products in these cells and, presumably, in vivo.

Stable expression of human cytochrome P450 1A1 cDNA in V79 Chinese hamster cells and metabolic activation of benzo[a]pyrene.

A V79 Chinese hamster cell line stably expressing human cytochrome P450 1A1 (CYP1A1) was obtained by chromosomal integration of the human CYP1A1 cDNA under the control of the SV40 early promoter. Chromosomal integration was verified by Southern analysis, and effective transcription of the human CYP1A1 cDNA was demonstrated by Northern analysis. The CYP1A1 cDNA-encoded protein was characterized by Western analysis using anti-rat CYP1A1. Intracellular association of CYP1A1 with the endoplasmic reticulum could be visualized by in situ immunofluorescence. Crude cell lysates of the V79 derived cell line was able to catalyze 7-ethoxyresorufin-O-deethylation (EROD) with an activity of about 50 pmol min-1 mg-1 total protein, and an aryl hydrocarbon hydroxylase activity (AHH) of 25 pmol min-1 mg-1. CYP1A1 dependent cytotoxicity, measured by neutral red uptake, and genotoxicity, determined by the frequency of micronucleus formation, of benzo[a]pyrene (B[a]P) and trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-diol) could be demonstrated at substrate concentrations as low as 10 nM. Thus, this cell line presents a sensitive tool for studying CYP1A1 mediated metabolism of polycyclic aromatic hydrocarbons (PAH). B[a]P and the purified (+)- and (-)-enantiomers of B[a]P-7,8-diol were compared for their mutagenicity. The (-)-enantiomer was found to be 3-5-fold more mutagenic than the (+)-enantiomer.

Genetically engineered V79 Chinese hamster cells for stable expression of human cytochrome P450IA2.

V79 Chinese hamster cells were genetically engineered for stable expression of human P450IA2. Full length cDNA, encoding human P450IA2, was inserted into an SV40 early promoter containing eukaryotic expression vector and cointroduced with the selection marker neomycin phosphotransferase (conferring resistance to the neomycin derivative G418) into V79 Chinese hamster cells. The recombinant expression vector was introduced into two different V79 sublines, one expressing an endogenous acetyltransferase (V79-NH), the other not (V79-MZ). The presence of human cytochrome CYP1A2 cDNA in the G418 resistant V79 cell clones was confirmed by Southern blotting. The transcription of the cDNA into mRNA was detected by Northern blotting and the translation into an authentic cytochrome P450IA2 protein was shown by Western blotting. The enzymatic activity in these cells was determined by the cytochrome P450IA2-dependent methoxy-, ethoxy-, benzoxy-, and pentoxyresorufin dealkylation activity.

Differential effects of 12-O-tetradecanoylphorbol 13-acetate on cytochrome P-450-dependent monooxygenase activities in rat hepatoma cells: induction of P-450I and suppression of P-450II.

We have studied the effects of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) on cytochrome P-450-dependent monooxygenase activities in several differentiated and dedifferentiated Reuber rat hepatoma cell lines using aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH), ethoxyresorufin O-deethylase (EROD), and aldrin epoxidase (AE) as test systems. The following results were obtained: (1) Exposure of cultures to 400 nM TPA for 18-24 h increased AHH activities in the differentiated lines 2sFou, H41IEC3/G- and Fao as well as in the dedifferentiated line 5L, 1.5-2.5-fold. The phorbol ester did not affect AHH activity in the dedifferentiated line H5. (2) EROD, a marker for P-450I, was induced by the phorbol ester to a similar degree as AHH. (3) A monoclonal antibody directed against P-450I strongly inhibited the AHH activity induced by TPA. (4) The onset of AHH or EROD induction by TPA was much later than that elicited by benz[a]anthracene. (6) In contrast to the induction of AHH and EROD, TPA decreased AE activity, a marker for P-450II, by about 50% in all the cell lines containing this monooxygenase activity. (7) The half-maximum-effect concentration of TPA for inducing or suppressing AHH and AE, respectively, was approximately 20 nM. (8) TPA did not interfere with AHH induction by benz[a]anthracene. However, the phorbol ester moderately decreased AHH induction and markedly suppressed AE induction by dexamethasone. The results indicate that TPA simultaneously induces P-450I and suppresses P-450II forms in rat hepatoma cells. P-450I induction by TPA in these cells did not appear to depend on their status of differentiation. Furthermore, the results suggest that the mechanism of P-450I induction by TPA differs from that elicited by polycyclic aromatic hydrocarbons or glucocorticoids.

Biological activity of 2,4,8-trichlorodibenzofuran: promotion of rat liver foci and induction of cytochrome P-450-dependent monooxygenases.

The biological activity of 2,4,8-trichlorodibenzofuran (2,4,8-TCDF) was tested using 2 endpoints: (a) the promotion of enzyme-altered, preneoplastic foci initiated by diethylnitrosamine (DEN) in livers of weanling female Sprague-Dawley rats; and (b) the induction of aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH), a marker for cytochrome P-4501 activity, in livers of adult female Sprague-Dawley rats and in H4IIEC3 rat hepatoma cells. When animals were treated with 200 or 500 mg/kg 2,4,8-TCDF 5 X weekly over 10 weeks after a single application of 10 mg/kg DNA, the higher dose of 2,4,8-TCDF had a promoting effect on the appearance of preneoplastic foci. Thus number and total area of foci deficient in adenosine-5'-triphosphatase were significantly increased by a factor of 1.6. 2,4,8-TCDF induced AHH-activities in 9000 X g supernatants of liver 2-3-fold, when rats were treated with 100-1000 mg/kg/day for 5 days and monooxygenase activities determined after another 3 days. The amounts of 2,4,8-TCDF required for inducing AHH activity in H4IIEC3 cells were 7 orders of magnitude higher than those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD). the results indicate that the 2,4,8-TCDF has a biological activity which is extremely low compared to that of 2,3,7,8-TCDD.

UDP-glucuronosyl-, phenol sulfo-, and glutathione-S-transferase activities of mammalian cells in permanent culture.

Established cell cultures derived from mouse, rat, hamster, cat, and man were examined for the in vitro activities of UDP-glucuronosyl-, phenol sulfo-, and glutathione-S-transferases. The relative activities of the conjugation reactions differed considerably between the various cell lines: Glutathione-S-transferase activities were present in all cell lines with wide variation between different transferase forms. Phenol sulfotransferase was not detectable at all in most of the cell lines tested or was present at very low levels. In contrast, UDP-glucuronosyltransferase activity was expressed in the majority of cultures. Established cultures containing specific combinations of the various types of conjugases should be useful in examining their role in the inactivation and activation of potentially toxic chemicals.

Dexamethasone-mediated potentiation of P450IA1 induction in H4IIEC3/T hepatoma cells is dependent on a time-consuming process and associated with induction of the Ah receptor.

The synergistic effect of dexamethasone (DEX) and polycyclic aromatic hydrocarbons on the induction of cytochrome P450IA1 (P450IA1) was examined in H4IIEC3/T Reuber hepatoma cells. P450IA1 activity was determined by the hydroxylation of benzo[a]pyrene (AHH) and deethylation of 7-ethoxyresorufin (EROD). The amount of Ah receptor, i.e. the specific cytosolic binding protein of 3-methylcholanthrene or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in H4IIEC3/T cells was characterized and quantitated by high performance gel filtration. Benz[a]anthracene and TCDD induced AHH and EROD activities, respectively, about 20-fold within 4 h. The increase was about 100-fold when cells were pretreated with DEX. The glucocorticoid alone induced P450IA1 activities 3-4 fold. DEX elicited half maximum AHH induction at a concentration of 20 nM in the presence or absence of benz[a]anthracene. Maximal potentiation of AHH induction required treatment with DEX for at least 32 h prior to the exposure to benz[a]anthracene. Treatment of H4IIEC3/T cells with DEX for 20 h caused a 2-3-fold increase in the amount of Ah receptor. The results suggest that the synergistic effect of DEX and polycyclic aromatic hydrocarbons on P450IA1 induction involves a time-consuming process which may consist of the synthesis or modification of a factor, possibly the Ah receptor.

Phenobarbital induces cytochrome P-450- and cytochrome P-448-dependent monooxygenases in rat hepatoma cells.

The induction by phenobarbital (PB) of aldrin epoxidase (AE) and aryl hydrocarbon hydroxylase (AHH), markers of cytochrome P-450- and cytochrome P-448-dependent monooxygenases, was studied in cell lines derived from Reuber H35 rat hepatoma which differ widely in their degree of differentiation. The following results were obtained: (1) PB induced AE 2-6-fold and AHH 2-4-fold in the differentiated clones, Fao, 2sFou, and C2Rev7 during an exposure period of 72 h. The barbiturate increased AHH but not AE in the dedifferentiated clone H5, the poorly differentiated line H4IIEC3/T, and in the well differentiated line H4IIEC3/G-. (2) Continuous presence of the barbiturate was required for maintaining the induction of the two monooxygenase activities in C2Rev7 cells. (3) Maximum induction of AE was observed at a PB concentration of 1.5-3.0 mM. (4) The effects of 7,8-benzoflavone on AHH-activities induced by phenobarbital in C2Rev7 and H5 cells suggested that they are mediated by cytochrome P-450- and cytochrome P-448-dependent monooxygenase forms, respectively. Thus, the flavonoid had only a slight inhibitory effect on PB-induced AHH in C2Rev7 cells, but strongly inhibited PB-induced AHH in H5 cells. The induction of AE and of 7,8-benzoflavone-inhibitable AHH in 2sFou cells indicated that PB is capable of inducing cytochromes P-450 and cytochrome P-448 in the same cell.

Retinyl acetate modulation of cell growth kinetics and carcinogen--cellular interaction in mouse epidermal cell cultures.

Exposure of mouse epidermal cell cultures to beta-retinyl acetate (RA) affects a number of parameters presumed to be important in chemical carcinogenesis. (1) RA alters the course of differentiation of the epidermal cells in culture resulting in a reduced rate of cell death which normally follows cellular maturation during the first two weeks in culture. The extended life span of the cultures appeared due to prolonged survival of cells and not to increased growth rate since RA inhibited the rate of cellular proliferation. This inhibition took place only after completion of a full cell cycle in the presence of RA. (2) DNA repair in response to physical and chemical agents was quantitatively unaffected in the presence of RA. (3) The activity of constitutive aryl hydrocarbon hydroxylase (AHH) was slightly decreased after exposure to RA but the level of enzyme induced by benz[a]anthracene was strongly reduced to 20% of the controls. (4) In the presence of RA, binding of 7,12-dimethylbenz[a]anthracene to epidermal cell DNA was markedly decreased. In contrast, binding to cellular protein was significantly increased by the retinoid.

Assessment of membrane damage in continuous cultures of mammalian cells.

The present studies were aimed at evaluating procedures for assessing the effect of chemicals on the integrity of the plasma membrane in continuous cell cultures. The degree of membrane damage was monitored by determining the 'leakage' of alpha-[3H]aminoisobutyric acid ([3H]AIB) and [14C]deoxy-2-fluoro-D-glucose ([14C]FdG) from the prelabelled cells. These parameters were compared to the loss of lactate dehydrogenase (LDH) from the cells and the decrease in the intracellular level of K+. Triton X-100, sodium dodecylsulfate (SDS), phospholipase C and nystatin which are known to affect membranes by different mechanisms served as test agents. In parallel, we monitored the effects of the chemicals on the viability of the cells. The following results were obtained: (1) The two radioactive markers [3H]AIB and [14C]FdG were found to be suitable to probe for damages of the plasma membrane in a variety of continuous cell lines which differ widely in their phenotype, rate of growth and degree of differentiation. (2) The leakage of the two markers could conveniently be monitored by double labelling techniques. (3) The loss from the cells of the 3 markers of smaller molecular size, K+, [3H]AIB, [14C]FdG, differed considerably depending on the test agent used. (4) Intracellular K+ level and [3H]AIB leakage generally appeared to follow a similar pattern, whereas [14C]FdG leakage may have shown a distinctly different response. (5) The leakage of LDH was an insensitive indicator for membrane damage. (6) No clear relationship was detectable between a particular leakage pattern of the markers and the loss of cellular viability.

Arylamine N-acetyltransferase activities in cell lines of mouse, rat, hamster and man differing in their sensitivity to 1,6-dinitropyrene.

This study was aimed at monitoring N-acetyltransferase activities of continuous cell lines, which differ in their sensitivity to the toxic effects of nitroaromatic compounds. Transferase activities were measured toward the acetyl acceptors sulfamethazine and p-aminobenzoic acid in partially purified preparation of cytosols. Cell lines such as hamster V79, BHK, rat hepatoma H4IIEC3G- or fibroblast 208F, which are sensitive to 1,6-dinitropyrene (1,6-DNP), possess high transferase activities ranging from 120-270 nmol/min x mg protein. In contrast, human lung cells NCI-H322, mouse and rat hepatoma cells BW1J and H5, respectively, which are resistant to 1,6-DNP contain no or low transferase activity of less than 15 nmol/min x mg. There was no apparent correlation between 1,6-DNP sensitivity and acetyltransferase levels in a few cell lines, e.g. rat hepatoma HTC, 2sFou and 5L, which express intermediate transferase activities ranging from 25-50 nmol/min x mg protein. The results suggest that acetylation is an essential step in activating 1,6-DNP to toxic products in mammalian cells.

V79 Chinese hamster cells express cytochrome P-450 activity after simultaneous exposure to polycyclic aromatic hydrocarbons and aminophylline.

V79 Chinese hamster lung cells expressed low but significant aryl hydrocarbon hydroxylase activities when treated with an inducer of cytochrome P-450I, such as benz[alpha]anthracene or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), together with aminophylline. Inducibility by polycyclic aromatic hydrocarbons and inhibition by a specific monoclonal antibody indicated that the observed enzyme activity was mediated by cytochrome P-450I. Intact V79 cells pretreated with TCDD and aminophylline for 24 h metabolized benzo[alpha]pyrene to phenolic products which accumulated linearly in the growth medium for at least the same time period. Exposure of V79 cells to 10 microM benzo[alpha]pyrene and aminophylline for 72 h reduced subsequent cell growth by about 40%. The results demonstrate that V79 cells, under specific conditions, express PAH-inducible cytochrome P-450I and are capable of activating benzo[alpha]pyrene to cytotoxic products.

Glutathione and glutathione S-transferase activities of mammalian cells in culture.

Continuous cell cultures derived from various tissues of rat, mouse, hamster and man were assayed for their glutathione (GSH) content and glutathione S-transferase activities. GSH S-transferase activities were monitored toward the substrates 1-chloro-2,4-dinitro-benzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB) and 1,2-epoxy-3-(p-nitrophenoxy)propane (PO). All cell lines tested contained appreciable amounts of GSH ranging from 10 to 65 nmol/mg cellular protein. Likewise, all cell lines expressed GSH S-transferase activities. However, the various cell lines differed considerably in their relative transferase activities exhibiting some degree of species-specificity.