Chimeric mice with humanized livers: a unique tool for in vivo and in vitro enzyme induction studies.

We performed in vivo and in vitro studies to determine the induction of human cytochrome P450 (CYP) using chimeric mice with humanized liver (PXB-mice®) and human hepatocytes isolated from the PXB-mice (PXB-cells), which were derived from the same donor. For the in vivo study, PXB-mice were injected with 3-methylcholanthrene (3-MC, 2 or 20 mg/kg) or rifampicin (0.1 or 10 mg/kg) for four days. For the in vitro study, PXB-cells were incubated with 3-MC (10, 50, or 250 ng/mL) or with rifampicin (5 or 25 µg/mL). The CYP1A1 and 1A2, and CYP3A4 mRNA expression levels increased significantly in the PXB-mouse livers with 20 mg/kg of 3-MC (Cmax, 12.2 ng/mL), and 10 mg/kg rifampicin (Cmax, 6.9 µg/mL), respectively. The CYP1A1 mRNA expression level increased significantly in PXB-cells with 250 ng/mL of 3-MC, indicating lower sensitivity than in vivo. The CYP1A2 and CYP3A4 mRNA expression levels increased significantly with 50 ng/mL of 3-MC, and 5 µg/mL of rifampicin, respectively, which indicated that the sensitivities were similar between in vivo and in vitro studies. In conclusion, PXB-mice and PXB-cells provide a robust model as an intermediate between in vivo and in vitro human metabolic enzyme induction studies.

Highly selective bioactivation of 1- and 2-hydroxy-3-methylcholanthrene to mutagens by individual human and other mammalian sulphotransferases expressed in Salmonella typhimurium.

The benzylic alcohols 1- and 2-hydroxy-3-methylcholanthrene (OH-MC) are major primary metabolites of the carcinogen 3-methylcholanthrene (MC). We investigated them for mutagenicity in TA1538-derived Salmonella typhimurium strains expressing mammalian sulphotransferases (SULTs). 1-OH-MC was efficiently activated by human (h) SULT1B1 (2400 revertants/nmol), weakly activated by hSULT1C3 and hSULT2A1 (2-9 revertants/nmol), but not activated by the other hSULTs studied (1A2, 1A3, 1C2 and 1E1). Mouse, rat and dog SULT1B1 activated 1-OH-MC (8-100 revertants/nmol) with much lower efficiency than their human orthologue. The other isomer, 2-OH-MC, was activated to a potent mutagen by hSULT1A1 (4000-5400 revertants/nmol), weakly activated by hSULT1A2 or hSULT2A1 (1-12 revertants/nmol), but not activated by the other hSULTs. In contrast to their human orthologue, mouse, rat and dog SULT1A1 did not appreciably activate 2-OH-MC (<1 to 6 revertants/nmol), either. Instead, mouse and rat SULT1B1, unlike their human and canine orthologues, demonstrated some activation of 2-OH-MC (15-100 revertants/nmol). Docking analyses indicated that 1- and 2-OH-MC might bind to the active site of hSULT1A1 and hSULT1B1, but only for (S)-2-OH-MC/hSULT1A1 and (R)-1-OH-MC/hSULT1B1 with an orientation suitable for catalysis. Indeed, 1- and 2-OH-MC were potent inhibitors of the hSULT1A1-mediated sulphation of acetaminophen [concentration inhibiting the enzyme activity by 50% (IC50) 15 and 13nM, respectively]. This inhibition was weak with mouse, rat and dog SULT1A1 (IC50 ≥ 4 µM). Inhibition of the SULT1B1 enzymes was moderate, strongest for 1-OH-MC/hSULT1B1. In conclusion, this study provides examples for high selectivity of bioactivation of promutagens by an individual form of human SULT and for pronounced differences in activation capacity between orthologous SULTs from different mammalian species. These characteristics make the detection and evaluation of such mutagens extremely difficult, in particular as the critical form may even differ for positional isomers, such as 1- and 2-OH-MC. Moreover, the species-dependent differences will complicate the verification of in vitro results in animal studies.

Disruption of the gene for CYP1A2, which is expressed primarily in liver, leads to differential regulation of hepatic and pulmonary mouse CYP1A1 expression and augmented human CYP1A1 transcriptional activation in response to 3-methylcholanthrene in vivo.

The cytochrome P4501A (CYP1A) enzymes play important roles in the metabolic activation and detoxification of numerous environmental carcinogens, including polycyclic aromatic hydrocarbons (PAHs). In this study, we tested the hypothesis that hepatic CYP1A2 differentially regulates mouse hepatic and pulmonary CYP1A1 expression and suppresses transcriptional activation of human CYP1A1 (hCYP1A1) promoter in response to 3-methylcholanthrene (MC) in vivo. Administration of wild-type (WT) (C57BL/6J) or Cyp1a2-null mice with a single dose of MC (100 µmol/kg i.p.) caused significant increases in hepatic CYP1A1/1A2 activities, apoprotein content, and mRNA levels 1 day after carcinogen withdrawal compared with vehicle-treated controls. The induction persisted in the WT, but not Cyp1a2-null, animals, for up to 15 days. In the lung, MC caused persistent CYP1A1 induction for up to 8 days in both genotypes, with Cyp1a2-null mice displaying a greater extent of CYP1A1 expression. It is noteworthy that MC caused significant augmentation of human CYP1A1 promoter activation in transgenic mice expressing the hCYP1A1 and the reporter luciferase gene on a Cyp1a2-null background, compared with transgenic mice on the WT background. In contrast, the mouse endogenous hepatic, but not pulmonary, persistent CYP1A1 expression was repressed by MC in the hCYP1A1-Cyp1a2-null mice. Liquid chromatography-mass spectrometry experiments showed that CYP1A2 catalyzed the formation of 1-hydroxy-3-MC and/or 2-hydroxy-3-MC, a metabolite that may contribute to the regulation of CYP1A1 expression. In conclusion, the results suggest that CYP1A2 plays a pivotal role in the regulation of hepatic and pulmonary CYP1A1 by PAHs, a phenomenon that potentially has important implications for PAH-mediated carcinogenesis.

Glucuronidation and sulfonation, in vitro, of the major endocrine-active metabolites of methoxychlor in the channel catfish, Ictalurus punctatus, and induction following treatment with 3-methylcholanthrene.

The organochlorine pesticide, methoxychlor (MXC), is metabolized in animals to phenolic mono- and bis-demethylated metabolites (OH-MXC and HPTE, respectively) that interact with estrogen receptors and may be endocrine disruptors. The phase II detoxication of these compounds will influence the duration of action of the estrogenic metabolites, but has not been investigated extensively. In this study, the glucuronidation and sulfonation of OH-MXC and HPTE were investigated in subcellular fractions of liver and intestine from untreated, MXC-treated and 3-methylcholanthrene (3-MC)-treated channel catfish, Ictalurus punctatus. MXC-treated fish were given i.p. injections of 2mg MXC/kg daily for 6 days and sacrificed 24h after the last dose. The 3-MC treatment was a single 10mg/kg i.p. dose 5 days prior to sacrifice. In hepatic microsomes from control fish, the V(max) value (mean+/-S.D., n=4) for glucuronidation of OH-MXC was 270+/-50pmol/min/mg protein, higher than found for HPTE (110+/-20pmol/min/mg protein). For each substrate, the V(max) values observed in intestinal microsomes were approximately twice those found in the liver. The K(m) values for OH-MXC and HPTE glucuronidation in control liver were not significantly different and were 0.32+/-0.04mM for OH-MXC and 0.26+/-0.06mM for HPTE. The K(m) for the co-substrate, UDPGA, was higher in liver (0.28+/-0.09mM) than intestine (0.04+/-0.02mM). Treatment with 3-MC but not MXC increased the V(max) for glucuronidation in liver and intestine. Glucuronidation was a more efficient pathway than sulfonation for both substrates, in both tissues. The V(max) values for sulfonation of OH-MXC and HPTE, respectively, in liver cytosol were 7+/-3 and 17+/-4pmol/min/mg protein and in intestinal cytosol were 13+/-3 and 30+/-5pmol/min/mg protein. Treatment with 3-MC but not MXC increased rates of sulfonation of OH-MXC and HPTE and the model substrate, 3-hydroxy-benzo(a)pyrene in both intestine and liver. Comparison of the kinetics of the conjugation pathways with those published for the demethylation of MXC showed that formation of the endocrine-active metabolites was more efficient than either conjugation pathway. Residues of OH-MXC and HPTE were detected in extracts of liver microsomes from MXC-treated fish. This work showed that although OH-MXC and HPTE could be eliminated by glucuronidation and sulfonation, the phase II pathways were less efficient than the phase I pathway leading to formation of these endocrine-active metabolites.

Antagonistic combinations of occupational carcinogens.

Several epidemiological and experimental studies demonstrate that combinations of carcinogens may interact in a synergistic way. This has prompted speculations that modulating interactions of individual chemical carcinogens are synergistic as a rule. However, various combinations of chemical carcinogens have been described which interact not even additively but in an antagonistic way. The aim of this review is to collect information of antagonistic interactions of occupational carcinogens obtained by epidemiologic and animal studies. In addition, appropriate in vitro studies with the genotoxic endpoints DNA-adducts and micronuclei are included. The toxicological mechanisms of antagonistic interactions, although speculative in some cases, are discussed.

Interleukin-1alpha and tumor necrosis factor alpha modulate cytochrome P450 activities in carp (Cyprinus carpio).

In mammals, it has been shown that the activation of host defense mechanisms down-regulates microsomal cytochrome P450 by the liberation of cytokines. We investigated the effect of interleukin-1alpha (IL1alpha) and tumor necrosis factoralpha (TNFalpha) on constitutive and 3-methylcholanthrene (3-MC)-induced biotransformation activities in carp. We have first measured the time course response of ethoxyresorufine O-decthylase (EROD) activity in liver, head kidney, and spleen 1, 2, 3, 5, and 7 days after intraperitoneal injection of a prototypical Cyp 1A inducer (3-MC). This activity was compared to the rate of 3-MC accumulation in all organs tested. A correlation between a diminution of EROD activity and an increase in 3-MC concentration in each organ was observed. We have also tested the effects of two inflammatory cytokines (IL1alpha and TNFalpha) on biotransformation activities. Intravenous injection of these compounds resulted in a marked depression of 3-MC-induced glutathione S-transferase activity in all organs tested and in 3-MC-increased cytochrome P450 content in the liver and head kidney. TNFalpha produced an increase in basal EROD activity in the liver and head kidney. Taken together, these results suggested that, as in mammals, the activation of host defense mechanisms regulates microsomal cytochrome P450 and related enzymes in fish.

Induction of Cyp1a1 and Cyp1b1 and formation of DNA adducts in C57BL/6, Balb/c, and F1 mice following in utero exposure to 3-methylcholanthrene.

Fetal mice are more sensitive to chemical carcinogens than are adults. Previous studies from our laboratory demonstrated differences in the mutational spectrum induced in the Ki-ras gene from lung tumors isolated from [D2 x B6D2F1]F2 mice and Balb/c mice treated in utero with 3-methylcholanthrene (MC). We thus determined if differences in metabolism, adduct formation, or adduct repair influence strain-specific responses to transplacental MC exposure in C57BL/6 (B6), Balb/c (BC), and reciprocal F1 crosses between these two strains of mice. The induction of Cyp1a1 and Cyp1b1 in fetal lung and liver tissue was determined by quantitative fluorescent real-time PCR. MC treatment caused maximal induction of Cyp1a1 and Cyp1b1 RNA 2-8 h after injection in both organs. RNA levels for both genes then declined in both fetal organs, but a small biphasic, secondary increase in Cyp1a1 was observed specifically in the fetal lung 24-48 h after MC exposure in all four strains. Cyp1a1 induction by MC at 4 h was 2-5 times greater in fetal liver (7000- to 16,000-fold) than fetal lung (2000- to 6000-fold). Cyp1b1 induction in both fetal lung and liver was similar and much lower than that observed for Cyp1a1, with induction ratios of 8- to 18-fold in fetal lung and 10- to 20-fold in fetal liver. The overall kinetics and patterns of induction were thus very similar across the four strains of mice. The only significant strain-specific effect appeared to be the relatively poor induction of Cyp1b1 in the parental strain of B6 mice, especially in fetal lung tissue. We also measured the levels of MC adducts and their disappearance from lung tissue by the P(32) post-labeling assay on gestation days 18 and 19 and postnatal days 1, 4, 11, and 18. Few differences were seen between the different strains of mice; the parental strain of B6 mice had nominally higher levels of DNA adducts 2 (gestation day 19) and 4 (postnatal day 1) days after injection, although this was not statistically significant. These results indicate that differences in Phase I metabolism of MC and formation of MC-DNA adducts are unlikely to account for the marked differences observed in the Ki-ras mutational spectrum seen in previous studies. Further, the results suggest that other genetic factors may interact with chemical carcinogens in determining individual susceptibility to these agents during development.

The effects of 3-methylcholanthrene on lymphocyte proliferation in the common carp (Cyprinus carpio L.).

The sensitivity of lymphocyte proliferation as bioindicator of pollution stress was evaluated in the common carp (Cyrinus carpio L.). The time course response of peripheral blood leukocyte proliferation in response or not to mitogens was measured from 1 to 7 days after peritoneal injection of 3-methylcholantrene (3-MC), and compared to the time course response of a highly sensitive biomarker, induction of cytochrome P450. 3-Methylcholanthrene (40 mg kg(-1)) inhibited both B- and T-lymphocyte proliferation in response to lipopolysaccharide (LPS) and concanavalin A (Con A). Studies with alpha-naphtofiavone, suggest the lack of metabolic processes. 3-Methylcholanthrene alone strongly stimulated resting peripheral blood leukocytes (PBLs) proliferation. This effect was not transient. The induction of lymphocyte proliferation paralleled the increase in cytochrome P450 content in the liver. The specificity of polycyclic aromatic hydrocarbon (PAH)-induced lymphocyte proliferation suggests that this immune activity may be an early marker of exposure to PAHs in aquatic environments. The capacity of 3-MC to induce rapid lymphocyte proliferation may be related to PAH-induced rapid clonal expansion in mammals. These results strongly suggested that the underlying mechanism might be the same in both models. More studies are needed in fish to explain this phenomenon and may be helpful in understanding the occurrence of neoplastic epizootics in fish associated with PAH exposition.

Persistent expression of 3-methylcholanthrene-inducible cytochromes P4501A in rat hepatic and extrahepatic tissues.

We reported earlier that 3-methylcholanthrene (MC) persistently induces hepatic ethoxyresorufin O-deethylase activities (CYP1A1) in rats for up to 45 days. In this investigation, we tested the hypotheses that persistent expression of CYP1A1 activities is paralleled by sustained induction of CYP1A1/CYP1A2 apoproteins and their mRNAs and that this phenomenon is mediated by mechanisms other than retention of MC in the rat. Rats were given MC (93 micromol/kg) i.p., once daily for 4 days, and CYP1A1/1A2 parameters were measured in liver at selected time points. MC-elicited increases in CYP1A1/1A2 activities, apoprotein contents, and mRNA levels were sustained for several weeks after the last dose of MC treatment. MC also caused long-term induction of CYP1A1 in lungs and mammary glands. Rats treated with [(3)H]MC once daily for 4 days excreted 92. 3% of the administered radioactivity in feces and urine by day 15. The intrahepatic concentration of MC at the 15-day time point was 270 pmol/g. Dose-response studies showed that administration of MC (2 micromol/kg), which produced an intrahepatic concentration of 271 pmol/g after 24 h, did not induce CYP1A1/1A2 activities, strongly suggesting that the sustained induction of CYP1A1/1A2 was not due to retention of the parent MC in the body. Electrophoretic mobility shift assays revealed that persistent CYP1A1 induction by MC involved Ah receptor-independent mechanisms. In conclusion, our results support the hypothesis that persistent expression of CYP1A1/1A2 by MC is mediated by mechanisms independent of the retention of the parent carcinogen.

Characterization of metabolites of benz(j)aceanthrylene in faeces, urine and bile from rat.

1. The excretion of benz[j]aceanthrylene (B[j]A) and the biotransformation products found in faeces, urine and bile of rat exposed to [3H]-labelled B[j]A have been studied. 2. About 95% of the administered radioactivity was excreted within 7 days, 79% via faeces and 16% via urine, and most of the radioactivity in urine and faeces was excreted within 2 days. 3. The B[j]A metabolites excreted between days 1 and 2, including those excreted in bile during the first 5.5 h in a separate experiment, were further characterized by HPLC, UV and electrospray/atmospheric pressure chemical ionization mass spectrometry. 4. In faeces, bile and urine, hydroxylated B[j]A metabolites predominated. The major metabolites in faeces were B[j]A-1,2-dihydrodiol-8-hydroxy and B[j]A-1,2-dihydrodiol-10-hydroxy. These metabolites were found as conjugated metabolites in the bile. The glucuronide conjugate of B[j]A-1,2-dihydrodiol-10-hydroxy was also a major metabolite in urine. Two sulphate conjugates of oxidized B[j]A were detected in bile, a sulphate conjugate of a B[j]A-dihydrodiol-phenol and B[j]A-1,2-dihydrodiol-10-sulphate. Trans-B[j]A-1,2-dihydrodiol was detected in urine, faeces and bile. 5. These findings support the hypothesis that epoxidation at the cyclopenta ring is an important biotransformation pathway for B[j]A in vivo. In addition to the characterized metabolites, a large fraction of polar compounds, possibly glutathione conjugates, was also excreted in urine and bile.

Polycyclic aromatic hydrocarbons enhance terminal cell death of human ectocervical cells.

Polycyclic aromatic hydrocarbons (PAH) are a class of chemical carcinogens whose active metabolites form DNA adducts, resulting in specific mutational events. The tumor suppressor protein p53 is believed to play a pivotal role in the ability of cells to response to DNA damage, resulting in either cell cycle arrest in G1 or apoptosis under conditions of excessive damage. This growth inhibition is associated with the concomitant induction of p53 and enhanced terminal cell differentiation. In this study we evaluated the effects of PAH on cell growth, cell differentiation, xenobiotic metabolism, and DNA adduct levels in normal ectocervical epithelial cells (ECE) and compared them to cervical cells whose p53 have been inactivated either by binding to viral HPV E6 oncogene (ECE16-1) or by mutation (C33A). The PAH 3-methylcholanthrene (3MC) inhibited normal ECE and to a lesser extent ECE16-1 cell proliferation. Not only did the growth inhibition occur at lower concentrations in the normal cells but the extent of inhibition was also greater in normal as compared to immortalized cells. Benzanthracene (BA) had a minor effect on normal ECE cells with no effect on immortalized ECE16-1 cells. C33A cell growth was unaffected by 3MC and BA. Terminal cell death was enhanced only in normal ECE cells as evidenced by increased envelope formation and was paralleled by an increase in the level of p53 following 3MC treatment. The differentiation status of the 3MC-treated cells was similar to untreated cells as indicated by three independent markers of cell differentiation; transglutaminase, involucrin, keratin expression. There was no difference in the pattern or level of DNA adducts formed in normal and immortalized cells following 3MC treatment. In addition the basal level of metabolism of 14C-BaP to phenols, diols and quinnones was unaltered by pretreatment with either 3MC or BA. These results demonstrate that immortalized cervical cells are less sensitive to toxicant damage [i.e. cell proliferation and terminal differentiation], and as a result, immortalized cells proliferate in the presence of genotoxic damage and are at increased risk for mutations and cancer.

Ki-ras mutations are an early event and correlate with tumor stage in transplacentally-induced murine lung tumors.

A previous study from this laboratory demonstrated that treatment of pregnant mice with 3-methylcholanthrene (MC) caused lung tumors in the offspring at 1 year after birth, the incidence of which correlated with fetal inducibility of Cyp1a1. Analysis by PCR amplification and allele-specific hybridization (ASO) of paraffin-embedded tumors generated from that study revealed the presence of point mutations in exon 1 of the Ki-ras gene. This work has now been expanded by PCR amplification and ASO analysis of 31 additional lesions. Point mutations were found in 37 of the 47 (79%) lesions analyzed in this and the previous study, the majority of which were G-->T transversions in the first or second base of codon 12. The mutational spectrum appeared to be dependent on the relative stage of differentiation of the lesion, as both the incidence of mutation and type of mutation produced correlated with malignant progression. Mutations occurred in 60% of the hyperplasias, 80% of the adenomas and 100% of the adenocarcinomas. In the lesions with mutations, GLY12-->CYS12 transversions occurred in 100% of the hyperplasias, 42% of the adenomas and 14% of the adenocarcinomas. The GLY12-->VAL12 transversions occurred in none of the hyperplasias, 42% of the adenomas and 57% of the adenocarcinomas. The remaining mutations, which consisted of ASP12 transitions and ARG13 transversions, occurred only in adenomas (17%) and adenocarcinomas (29%). Between this study and our previous analyses, the identity of the mutations obtained by ASO were confirmed by sequence analysis of eight of the 37 lesions that harbored mutations at the Ki-ras gene locus. There were no differences in the type or incidence of mutations relative to the metabolic phenotype or sex of the mice. These data suggest that mutational activation of the Ki-ras gene locus is an early event in transplacental lung tumorigenesis, and that the type of mutations produced by exposure to chemical carcinogens can influence the carcinogenic potential of the tumor. This may have prognostic significance in determining the malignant progression of the neoplasm.

Uptake and efflux of polycyclic aromatic hydrocarbons by Tetrahymena pyriformis: evidence for a resistance mechanism.

The action of benzo(a)pyrene (BP), 3-methylcholanthrene (3MC), benzanthracene (BA), and 7,12-dimethylbenzanthracene (DMBA), four polycyclic aromatic hydrocarbons (PAHs), was studied on the unicellular protozoan Tetrahymena pyriformis. This ciliate was exposed to the PAHs at 1, 15, and 37 microM for up to 6 h. BP and BA caused a slight inhibition of cell growth, whereas 3MC and DMBA showed no detectable effect. Cell viability remained unaffected by the PAHs at all concentrations and exposure times tested. Cellular accumulation of PAHs was studied using flow cytometry. The results show immediate accumulation followed by rapid elimination of the compounds. BP uptake was also studied in the presence of verapamil and cyclosporin, compounds known as inhibitors of the multidrug resistance (MDR) pump. In the presence of verapamil, BP was accumulated in larger amounts in cells. With cyclosporin, the accumulation of the PAH was several times higher than under control conditions. The results of GC/MS analysis show that PAH elimination was not linked to biotransformation. These results suggest that the resistance of Tetrahymena against PAH cytotoxicity may be attributed to the rapid efflux of these agents from the cells via an efflux pump probably of the MDR type.

Intrinsic mutagenicity and electrophilicity of 1-sulfooxy-3-methylcholanthrene: implications for metabolic activation of the carcinogen 3-methylcholanthrene.

Hydroxylation of a meso-anthracenic carbon atom with subsequent formation of a reactive ester bearing a good leaving group (e.g., sulfate) has been proposed as a possible biochemical mechanism responsible for DNA binding, mutagenicity and tumorigenicity of 3-methylcholanthrene, one of the most potent carcinogenic polycyclic aromatic hydrocarbons in experimental animals. In support of this supposition, the chemically synthesized sulfuric acid ester, 1-sulfooxy-3-methylcholanthrene (1-SMC) was directly mutagenic in bacteria and covalently bound to DNA without metabolic activation. The intrinsic mutagenicity of this reactive ester was significantly potentiated by addition of extra acetate or chloride anions to the media. Reduced glutathione and ascorbic acid protected against 1-SMC-induced mutagenesis. These findings suggest 1-SMC as a potential ultimate electrophilic and tumorigenic metabolite of 3-methylcholanthrene.

32P-postlabelling in studies of polycyclic aromatic hydrocarbon activation.

Polycyclic aromatic hydrocarbons (PAHs) undergo metabolic activation reactions to yield intermediates that react with DNA to form covalent adducts. PAH administration leads to the formation of various types of DNA adducts that may differ between species, strains and tissues due to differences in metabolic activation and repair. The structures of PAH-DNA adducts can be identified by three approaches: co-chromatography with synthetic mononucleotide adduct standards; examining the adducts resulting from metabolism of pathway intermediates; or chemically blocking metabolic activation at specific sites on the PAH. Administration of putative metabolic intermediates of a PAH leads to enhanced formation of DNA adducts resulting from further activation along that pathway. Conversely, chemically blocking a bond or position on a PAH prevents adducts arising from activation at that site. By comparing the DNA adduct spectra generated by metabolites, blocked forms, and parent PAH administration, the pathways important in the metabolic activation of the PAH in each tissue may be deduced. Partial identification of these adducts may also be made by co-chromatography with the products of reactions between synthetic reactive intermediates and defined polydeoxynucleotides, and more thorough identification by using synthetic DNA adduct standards. These approaches have all been successfully applied to studies of PAH activation, and are reviewed here.

Genetic requirements for frameshift reversion induced by bulky DNA adducts in M13 DNA.

In order to analyze the genetic requirements and mechanisms of frameshift mutagenesis by activated aflatoxin B1 (AFB1), in vitro-modified phage M13 replicative form (RF) DNA was transfected into appropriate Escherichia coli cells and +1 or -1 frameshift revertants in the lacZ(alpha) gene were isolated. This analysis shows that both +1 and -1 frameshift mutagenesis by AFB1 is significantly reduced in a umuC- background. On the other hand, in the absence of RecA, +1 frameshift mutagenesis is partially reduced, but -1 frameshift mutagenesis is unaffected. DNA sequence analysis of +1 frameshifts induced by AFB1 in recA- cells suggests that the mutations occur at the same sites as in recA+ cells, but that there are significant differences in the specificity of the observed base changes. A model consistent with the observed effects in the absence of RecA suggests that an appreciable fraction of AFB1-adducted guanines can correctly template for a cytosine.

Possible mechanism for the cocarcinogenic effect of bile acids: increased intracellular uptake of methylcholanthrene by C3H/10T1/2 fibroblasts in vitro.

The cocarcinogenic effect of bile acids on the chemical transformation of C3H/10T1/2 fibroblasts was examined in vitro. The assimilation of 3H-methylcholanthrene (3H-MCA) by C3H/10T1/2 cells pretreated with bile acids was examined by the measurement of uptake and by autoradiography. Cells that were pretreated for 48 h with bile acids (100 microM lithocholic acid, 500 microM cholic acid) and then maintained in medium that contained 3H-MCA showed an increase in radioactivity compared to control cells. These results indicate that the transfer of carcinogens into cells is enhanced by pretreatment with bile acids.