Mutations induced by benzo [a] pyrene diolepoxide at the< i> hprt locus in human T-lymphocytes in vitro (original) (raw)
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Mutations induced by benzo[a]pyrene diolepoxide at the hprt locus in human T-lymphocytes in vitro
Mutation Research Letters, 1990
Human T-lymphocytes have been treated with benzo[a]pyrene diolepoxide (BPDE) in vitro and T-cell clones mutated in the hprt gene have been isolated. The mutant frequencies in BPDE-treated T-cell cultures were on average 24-fold higher than those of untreated cultures. Thus, BPDE is a potent inducer of gene mutation in this system. In order to examine which types of mutations are induced by BPDE in human cells, 41 spontaneous and 44 BPDE-induced mutant clones have been characterized using the Southern blot technique. In addition, rearrangements of the T-cell-receptor fl and 7 loci have been used to determine the proportion of isolated clones that are unique, and thus likely to represent independent mutational events. Out of 23 independent spontaneous mutants 4 had large hprt alterations that could be detected on Southern blots. Two of these alterations, deletions of exons 2-6, have been confirmed using PCR of hprt cDNA and direct sequencing of the PCR product. All 33 independent BPDE-induced mutants had normal hprt restriction patterns which indicates that BPDE is mainly a point mutagen in this system. Polycyclic aromatic hydrocarbons (PAH's) are common in the environment and include many mutagenic and carcinogenic compounds . The best studied PAH is benzo[a]pyrene. In mammalian cells, benzo[a]pyrene is metabolized to a variety of different compounds including 7,8-di-
Mutational spectra vary with exposure conditions: benzo[a]pyrene in human cells
Mutation research, 1996
The AHH-1 human lymphoblastoid line was exposed to benzo[a]pyrene under markedly different conditions: a single toxic exposure of 30 microM for 28 h, a nontoxic exposure of 0.5 microM for 6 days and an exposure approximating estimates of BP concentration in the human lung of 20 nM for 20 days. Duplicate cultures containing 2 x 10(9) cells each were used to assure the statistical quality of the mutational spectra. Point mutational hotspots were observed in bp 215 to 318 of the third exon of the hprt gene after mutants were selected en masse with 6-thioguanine, using a combination of denaturing gradient gel electrophoresis and high fidelity polymerase chain reaction. The spectra were highly reproducible in replicate experiments but varied dramatically among treatment conditions. These data demonstrate that mutational spectra were critically dependent upon conditions of exposure. The results significantly extend prior reports on this subject and clarify an important issue for the use o...
Cancer research, 1984
Benzo(e)pyrene (BeP) is a cocarcinogen with benzo(a)pyrene (BaP) and an anticarcinogen with 7,12-dimethylbenz(a)anthracene (DMBA) in mouse skin initiation-promotion assays (Slaga, T.J., Jecker, L., Bracken, W.M. and Weeks C.E. Cancer Lett. 7: 51-59, 1979). We have investigated the effects of BeP on the metabolic activation of BaP and DMBA in early-passage cultures of Syrian hamster embryo cells. BeP had no effect on BaP-induced mutation frequencies in hamster embryo cell-mediated assays with V79 target cells. However, it inhibited the DMBA-induced mutagenesis by as much as 10-fold at the highest dose tested. Low doses of BeP did not affect the total amount of BaP metabolized, but the proportion of water-soluble metabolites was reduced, and the proportions of trans-7,8-dihydro-7,8-dihydroxybenzo(a)pyrene and trans-9,10-dihydro-9,10-dihydroxybenzo(a)pyrene were increased. Higher doses did decrease BaP metabolism and caused similar alterations in the metabolite profile. In cultures tre...
Repair of DNA damaged by mutagenic metabolites of benzo(a)pyrene in human cells
Chemico-Biological Interactions, 1978
The repair of human DNA after damage by known and potential metabolites of benzo(a)pyrene has been examined utilizing the bromodeoxyuridine photolysis assay. Repair was characterized as either ultraviolet ("long") or ionizing radiation type ("short") repair utilizing normal cells and cells deficient in ultraviolet-type repair endonuclease from a patient with xeroderma pigmentosum (XP). We have found that only (+-)-7~,8a~iihydroxy-9~,-10~poxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BP diol epoxide 1) and its disastereomer, (+-)-7~,Sa,~lihydroxy-9a,10~ ~poxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BP diol epoxide 2) elicit damage to DNA which is recognizable by the ultraviolet excision repair system in normal human cells. Benzo(a)pyrene 4,5-, 9,10-, ll,12~xides do not elicit damage which is repairable by this repair system. The 1,2<liol-3,4~poxides from naphthalene have no measurable activity in our assay. These results indicate that both the benzo(a)pyrene ring structure and the diol epoxide groups are important in causing the damage to DNA which is repairable by the ultraviolet excision repair system. These results parallel the reported high mutagenic activity of these compounds and support the concept that benzo(a)pyrene 7,8~liol-9,10epoxides may be the ultimate, metabolically activated forms of benzo(a)pyrene.
1994
4Sytotoxicity and mutagenicity were measured in human lymphocytes after treatment in vitro with anti-or syn-benzo[a]pyrene diolepoxide, two diastereoisomer metabolites of benzo[a]pyrene. These compounds were incubated with resting and cycling lymphocytes to determine the inhibition of cell proliferation induced by phytohemoagglutinin and interleukin2 at different times after treatment. Anti-benzo[a]pyrene diolepoxide was more cytotoxic than the syn-adduct under all conditions, and its effect on cell growth was more marked in cycling lymphocytes. In contrast, neither of the compounds induced alteration of the ATP intracellular pool. Cytotoxic effects of anti-and syn-benzo[a]pyrene diolepoxide were also assessed by determining the cloning etficiency. Both compounds affected the cloning efficiency in human lymphocytes and the effect of anti-benzo[a]pyrene was particularly marked. Mutagenic potency of anti-and syn-benzo[a]pyrene diolepoxide at the hgprt locus was measured both in the V79 cell line and in human lymphocytes by selection of mutant cells in medium containing 6-thioguanine. Both compounds increased the mutant frequency in comparison with the control and anti-benzo[a]pyrene diolepoxide was more active than the syn-metabolite.
Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2016
Benzo[a]pyrene (B[a]P) is a known genotoxicant and carcinogen, yet its genotoxic response at low level exposure has not been determined. This study was conducted to examine the interplay of dose and metabolic capacity on the genotoxicity of B[a]P. An improved understanding the biological significance of low level chemical exposures will help to improve human health risk assessments. The genotoxic and mutagenic effects of B[a]P were investigated using human cell lines AHH-1, MCL-5, TK6 and HepG2, with differential expression of the CYP450 enzymes (CYP1A1, 1B1 and1A2) involved in B[a]P metabolism. MCL-5 and HepG2 cells showed detectable basal expression and activity of CYP1A1, 1B1 and 1A2 as compared to AHH-1, which only show CYP1A1 basal expression and activity. TK6 cells showed negligible expression levels of all three CYP450 enzymes. In vitro micronucleus and the hypoxanthine phosphoribosyltransferase assays were conducted to determine the effect of B[a]P on chromosome damage and point mutation induction. After 24h exposure, concentration-dependent increases in micronucleus (MN) frequency were observed in all cell lines except TK6. After 4h exposure, only the metabolically competent cell lines MCL-5 and HepG2 showed MN induction (with a threshold concentration at 25.5µM from MCL-5 cells) indicating the importance of exposure time for genotoxicity. The HPRT assay also displayed concentration-dependent increases in mutant frequency in MCL-5 cells, after 4h and 24h treatments. Mutation spectra analysis of MCL-5 and AHH-1 HPRT mutants revealed frequent B[a]P induced G to T transversion mutations (72% and 44% of induced mutations in MCL-5 and AHH-1, respectively). This study therefore demonstrates a key link between metabolic capability, B[a]P exposure time and genotoxicity.
Chemical Research in Toxicology, 2007
Benzo[a]pyrene (B[a]P), a representative polycyclic aromatic hydrocarbon (PAH), is metabolically activated by three enzymatic pathways; by peroxidases (e.g. cytochrome P450-peroxidase) to yield radical cations; by P4501A1/1B1 monoxygenation plus epoxide hydrolase to yield diol-epoxides; and by P4501A1/1B1 monoxygenation, epoxide hydrolase plus aldo-keto reductases (AKRs) to yield o-quinones. In humans, a major exposure site for environmental and tobacco smoke PAH is the lung, however, the profile of B[a]P metabolites formed at this site has not been well characterized. In this study, human bronchoalveolar H358 cells were exposed to B[a]P, and metabolites generated by peroxidase (B[a]P-1,6-and B[a]P-3,6-diones), from cytochrome P4501A1/1B1 monooxygenation (3-hydroxyl-B[a]P, B[a]P-7,8-and 9,10-trans-dihydrodiols, and B[a]P -r-7,t-8,t-9,c-10tetrahydrotetrol (B[a]P -tetrol-1)), and from AKRs (B[a]P-7,8-dione) were detected and quantified by RP-HPLC-with in line photo-diode array and radiometric detection, and identified by LC-MS. Progress curves showed a lag-phase in the formation of 3-hydroxy-B[a]P, B[a]P-7,8-transdihydrodiol, B[a]P-tetraol-1 and B[a]P-7,8-dione over 24 h. Northern blot analysis showed that B
Cytotoxic and mutagenic effects of anti- and syn-benzo[a]pyrene diol epoxide in human lymphocytes
Toxicology in Vitro, 1994
4Sytotoxicity and mutagenicity were measured in human lymphocytes after treatment in vitro with anti-or syn-benzo[a]pyrene diolepoxide, two diastereoisomer metabolites of benzo[a]pyrene. These compounds were incubated with resting and cycling lymphocytes to determine the inhibition of cell proliferation induced by phytohemoagglutinin and interleukin2 at different times after treatment. Anti-benzo[a]pyrene diolepoxide was more cytotoxic than the syn-adduct under all conditions, and its effect on cell growth was more marked in cycling lymphocytes. In contrast, neither of the compounds induced alteration of the ATP intracellular pool. Cytotoxic effects of anti-and syn-benzo[a]pyrene diolepoxide were also assessed by determining the cloning etficiency. Both compounds affected the cloning efficiency in human lymphocytes and the effect of anti-benzo[a]pyrene was particularly marked. Mutagenic potency of anti-and syn-benzo[a]pyrene diolepoxide at the hgprt locus was measured both in the V79 cell line and in human lymphocytes by selection of mutant cells in medium containing 6-thioguanine. Both compounds increased the mutant frequency in comparison with the control and anti-benzo[a]pyrene diolepoxide was more active than the syn-metabolite.
Cancer Letters, 1986
Co-administration of benz [a] anthracene (BA) with benzo [a ] pyrene (B[a]P) to hamster embryo cell cultures for 24 h resulted in a decrease in the metabolism of benzo[a]pyrene by 40%, a decrease in the level of binding of B[a]P to DNA by 70% and a lo-fold reduction in mutation induction in a hamster embryo cell-mediated V79 cell mutation assay. This data indicates that the biological effects of co-administration of BA with B[a] P result from inhibition of the metabolic activation of B[a] P rather than induction of enzymes that detoxify the B[a] P.
Metabolism of benzo(a)pyrene by murine embryonal carcinoma cells
PubMed, 1981
Murine embryonal carcinoma (EC) cells were characterized with respect to their ability to metabolize the polycyclic aromatic hydrocarbon, benzo(a)pyrene [B(a)P]. The extent of metabolic activation varied more than 100-fold among the teratocarcinoma-derived cell lines examined. This difference in metabolic activity was correlated with an increase in the formation of specific metabolites that were identified by high-pressure liquid chromatography. Maximal in vitro formation of water-soluble products occurred 24 hr after the addition of [3H]B(a)P to the EC cells. Long-term incubation of EC cells with [3H]B(a)P indicated that, within the initial 24 hr, 2.3% of the input had been taken up by the cells. Subcellular analysis of the distribution of radioactivity indicated that 70 to 80% of intracellular radioactivity was associated with isolated nuclei. Therefore, the intranuclear metabolites of B(a)P were also analyzed by high-pressure liquid chromatography. Embryonal carcinoma cell lines OC15S1 and C86S1 showed significant in vitro toxic effects to B(a)P over a concentration range of 0.05 to 0.3 microgram/ml, whereas F9 and PC13 were resistant to concentrations of B(a)P up to 5 microgram/ml. Equally resistant to B(a)P was the PYS cell line, a differentiated cell type derived from EC cells. Cytotoxicity was related to the extent of metabolic activation of parent compound.