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Query: UNIPROT:P06889 (Mol)
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Our recent syntheses of chryseno[4,5-bcd]thiophene together with its potential sulfone metabolite, chryseno[4,5-bcd]thiophene-4,4-dioxide, have made these compounds available for genotoxicity testing. Such toxicity testing is of interest as this thiophene is an isoster of the established carcinogen benzo[a]pyrene and is one of the thiaarenes which are potential environmental contaminants found in fossil fuels. Although the thiophene was less mutagenic than benzo[a]pyrene in Salmonella strains TA98 and TA100 after S9 activation, it exhibited in vivo chromosomal aberration activity equal to that of benzo[a]pyrene in the bone-marrow cells of mice. A reduced activity with Salmonella as well as in the bone-marrow cell assay for the sulfone does not support its role as the key active metabolic intermediate for the genotoxicity of the thiophene. Our molecular orbital calculations would be consistent with the concept of activation through a diol-epoxide mechanism and offers an explanation for the reduced genotoxicity of the sulfone via this mechanism. These genotoxicity studies support the concern that sulfur isosters of established carcinogenic polycyclic aromatic hydrocarbons could themselves be toxic.
Environ Mol Mutagen 1992
PMID:Genotoxicity of chryseno[4,5-bcd]thiophene and its sulfone derivative. 157 49

Identification of tumor suppressor gene loci in rodent cell culture systems has relied upon the use of somatic cell hybridization studies. Although normal rodent fibroblasts are capable of suppressing the tumorigenicity of a variety of tumor cells, the lack of complementation in tumor cell x tumor cell hybrids has left the possibility that a single tumor suppressor gene may be responsible for tumor suppression in a particular rodent cell culture system. Using this same approach, we found no evidence for complementation resulting in suppression of the transformed phenotype when three viral oncogene-transformed Syrian hamster embryo (SHE) cell lines and one spontaneously transformed baby hamster kidney (BHK) cell line were fused to benzo[a]pyrene-transformed SHE cells (BP6T-M3). However, v-src oncogene-transformed cell line (srcT) x BP6T-M3 hybrids did demonstrate limited suppression of the transformed phenotype, suggesting at least two complementing tumor suppressor genes in this system. We were able to confirm and extend this finding using another experimental approach with preneoplastic hamster cell lines that are immortal in culture but nontumorigenic in nude mice. We propose that fusion of these preneoplastic cells to various tumor cells may reveal tumor suppressor genes not evident in the tumor cell x tumor cell complementation studies. Subclones of two nontumorigenic, immortal hamster cell lines, 10W and DES4, displayed differing abilities to suppress BP6T-M3 cells in somatic cell hybrids, as quantitated by the ability of the hybrid cells to form colonies in soft agar. With a panel of preneoplastic hamster cell x BP6T-M3 hybrids, a distinct pattern of suppression or expression of the transformed phenotype was observed. Marked differences in this pattern were seen when the same 10W and DES4 subclones were fused to other hamster fibrosarcoma cell lines, indicating different tumor suppressing activities of multiple tumor suppressor genes. Analysis of this data suggests that as few as three or as many as six different tumor suppressor genes may be active in the Syrian hamster embryo cell culture system. Thus, this system may provide a useful model for identifying and studying the effects and regulation of a number of different tumor suppressor genes for fibrosarcomas.
Somat Cell Mol Genet 1992 Mar
PMID:Detection of multiple tumor suppressor genes for Syrian hamster fibrosarcomas by somatic cell hybridization. 157 39

Rat and human lung microsomal cytochrome P-450 (P-450) enzymes have been characterized with regard to their catalytic activities towards several xenobiotic chemicals, including procarcinogens, in different microsomal preparations. Rat lung microsomal P-450s were more active than the human P-450s in catalyzing most of the monooxygenation reactions. Human lung microsomal P-450 was solubilized and purified. Human lung microsomes contain approximately 10 pmol of P-450/mg of protein, on the basis of Fe2+.CO versus Fe2+ difference spectra of the eluates obtained from an octylamino-agarose column. The partially purified P-450 preparations from two human lung microsomal samples showed high activities for the conversion of both (+)- and (-)-isomers of 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene to genotoxic products. After DEAE-cellulose column chromatography, a partially purified P-450 fraction containing polypeptides of Mr 52,000 and 58,000 was obtained from the early fraction of the octylamino-agarose column eluate, and an electrophoretically homogeneous protein having a molecular weight of approximately 52,000 was recovered from a latter fraction. The amino-terminal amino acid sequences of the two peptides in the earlier fraction were determined; neither polypeptide appears to resemble any known P-450 protein. The protein from the latter octylamino-agarose fraction was immunoreactive with anti-rat P-450 1A2 and anti-human P-450 1A2 but not with antibodies raised against other P-450 enzymes or autoimmune antibodies that specifically recognize human P-450 1A2. A tryptic peptide was isolated from the preparation, and the amino acid sequence matched that of human P-450 1A1 perfectly (residues 31-48) but not that of human P-450 1A2. All of nine human lung microsomal samples examined contained proteins that were immunoreactive with rabbit anti-rat P-450 1A2 and catalyzed the activation of 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene. The activities could be inhibited by rabbit anti-rat P-450 1A2 and, to a lesser extent, by anti-rat P-450 1A1. The addition of 7,8-benzoflavone caused inhibition or stimulation, depending upon the particular human lung microsomal preparation. Thus, this work clearly shows that human lung microsomes contain at least two major P-450 enzymes; human P-450 1A1 is present in lungs and can actually catalyze the activation of environmental procarcinogens, including polycyclic aromatic hydrocarbons.
Mol Pharmacol 1992 May
PMID:Characterization of human lung microsomal cytochrome P-450 1A1 and its role in the oxidation of chemical carcinogens. 158 20

The SOS chromotest was applied for the detection of antimutagens. To raise SOS induction, the bacteria were treated with the mutagens, UV, 4-nitroquinoline N-oxide (4NQO), N-methyl-N'-nitro-N-nitroso-guanidine (MNNG), or benzo[a]pyrene (B[a]p). The inhibitory effects of L-ascorbic acid, glutathione, vanillin, 5-fluorouracil (5-FU), 5-chlorouracil (5-CU), cobaltous chloride, sodium selenite and sodium arsenite, which are known as antimutagens, were investigated with their addition either simultaneously or post treatment time. It became clear that the SOS chromotest was very useful for the detection of antimutagens.
Environ Mol Mutagen 1991
PMID:Evaluation of the SOS chromotest for the detection of antimutagens. 164 15

The aromatic hydrocarbon (Ah) receptor mediates induction of cytochrome P4501A1 and associated aryl hydrocarbon hydroxylase (AHH) activity in tissues or cells exposed to polycyclic aromatic hydrocarbons. Strains of mice designated "nonresponsive" do not show increased hepatic AHH activity when exposed in vivo to nonhalogenated aromatic hydrocarbons such as 3-methylcholanthrene, benz[a]anthracene (BA), or benzo[a]pyrene and have reduced sensitivity to halogenated inducers such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Recently, with a modified assay, we detected Ah receptor in hepatic cytosols from adult nonresponsive mice [Mol. Pharmacol. 35:823-830 (1989)]; the receptor was present in reduced amount, and the apparent affinity for TCDD was lower than in hepatic cytosol from responsive C57BL/6J mice. Using the same assay procedure, we now report detection of Ah receptor in cytosols prepared from embryonic tissue and from cultured embryo cells of both responsive (C57BL/6J) and nonresponsive mice (DBA/2J, AKR/J, and SWR/J). Cytosolic receptor in embryonic cells from nonresponsive as well as responsive strains was detectable both with [3H]TCDD and with [3H]3-methylcholanthrene. In addition, the receptor-ligand complex could be extracted from nuclei of embryo cells exposed to [3H]TCDD in culture. AHH activity was induced in embryo cell cultures incubated with either TCDD or BA. The EC50 values for AHH induction were virtually identical in cell cultures from nonresponsive (DBA/2J) and responsive (C57BL/6J) strains, using either TCDD or BA as the inducer. Moreover, the affinity with which [3H]TCDD bound to cytosolic Ah receptor was much more similar in cytosols from cell cultures from the two strains than in cytosols prepared from adult liver. Thus, embryonic cell cultures differ in at least three respects from the adult liver, as follows: (i) Ah receptor can be detected with [3H]3-methylcholanthrene in embryonic cell cytosols but not in cytosols from adult liver; (ii) the degree of difference between nonresponsive and responsive strains in the affinity with which [3H]TCDD binds to receptor is only about 2-fold in cytosol from embryonic cells, whereas it is almost 10-fold in adult liver; and (iii) induction of AHH activity (by either TCDD or by the nonhalogenated inducer BA) shows no significant difference between strains in embryonic cell culture, whereas there is at least a 15-fold difference in responsiveness between C57BL/6J and DBA/2J mice in adult liver in vivo. The mechanistic reason for the diminished degree of difference between responsive and nonresponsive mice during embryonic cell culture (compared with adult tissues) is not yet known.
Mol Pharmacol 1991 Nov
PMID:Ah receptor in mice genetically "nonresponsive" for cytochrome P4501A1 induction: cytosolic Ah receptor, transformation to the nuclear binding state, and induction of aryl hydrocarbon hydroxylase by halogenated and nonhalogenated aromatic hydrocarbons in embryonic tissues and cells. 165 12

V79 Chinese hamster cells genetically engineered to express cytochrome P-450IA1 are reported. A full length cDNA encoding rat cytochrome P-450IA1 was obtained from a cDNA library prepared from rat liver mRNA. The cDNA was recombined with the SV40 early promoter and expressed in V79 cells. Three V79-derived P-450IA1-expressing cell lines (XEM1, XEM2, and XEM3) were established. The presence of the rat cytochrome P-450IA1 cDNA in these hamster cells was confirmed by Southern blotting. The transcription of the cDNA into mRNA and translation into the desired cytochrome P-450 protein was detected by Northern and Western blotting. The enzymatic activity was determined by the cytochrome P-450IA1-dependent oxidation of benzo[a]pyrene and 7-ethoxycoumarin. After exposure to benzo[a]pyrene, the mutant frequency increased in XEM1 and XEM2 cells and was higher than in V79 cells in the presence of an exogenous activating system. The mutant frequency was even more increased when XEM1 and XEM2 cells were exposed to the proximate mutagen (trans)-7,8-dihydroxy-7,8-dihydro-benzo[a]pyrene.
Mol Pharmacol 1990 May
PMID:Stable expression of rat cytochrome P-450IA1 cDNA in V79 Chinese hamster cells and their use in mutagenicity testing. 169 5

5-Azacytidine (AZC) was studied in a lung cancer model in outbred and syngeneic (F1D) hamsters wherein benzol[a]pyrene (BP) from sustained release implants (SRI) induces preneoplastic mucosal changes which progress to bronchogenic cancer. In pilot studies to evaluate AZC toxicity, a dose schedule of 5 mg/kg biweekly was found suitable and was then used for long-term administration in all subsequent studies. Three groups of outbred hamsters were studied: BP SRi alone (n = 60), BP SRI + AZC (n = 60), and AZC alone (n = 54). AZC treatment was begun 3-5 days after SRI placement. Sixty-one days after the start of the experiment, seven or eight hamsters were sacrificed from each group. Later sacrifices were at 3-week intervals in groups receiving BP SRI and at 6-week intervals in the AZC only group. Four groups of F1D syngeneic hamsters were studied: BP SRI alone (n = 50); BP + AZC starting 3-5 days after SRI placement and continuing until death (n = 52); BP + AZC from 3 to 5 days until 75 days after SRI placement (n = 49); BP + AZC starting 80 days after SRI placement and continuing until death (n = 52). Hamsters (n = 9-14) from each group were sacrificed at 120, 150, 180, and 220 days after SRI implantation. AZC alone was not carcinogenic under these conditions. Both outbred and F1D hamsters treated with early or continuous AZC had slower rates of neoplastic change from BP SRI than did animals receiving BP SRIs alone or BP + late AZC. The incidence of epidermoid cancer were the same for all regimens, but the tumors in those receiving AZC early in carcinogenesis were smaller than in those receiving late or no AZC. The incidences of nonepidermoid cancer were lower in those receiving AZC during early carcinogenesis, and larger tumors were noted in the absence of AZC. Thus, within the study period in this unique hamster lung cancer model, AZC given early in carcinogenesis inhibited only the later (promotional) phase of BP epidermoid carcinogenesis, but inhibited all phases of nonsquamous cancer development induced by BP. This differential modulation of bronchial carcinogenesis, which occurs from AZC given during preneoplastic stages, may prove useful for delineating molecular mechanisms underlying specific phenotypic types of bronchogenic cancers.
Exp Mol Pathol 1990 Aug
PMID:Effects of 5-azacytidine in Syrian golden hamsters: toxicity, tumorigenicity, and differential modulation of bronchial carcinogenesis. 169 60

Previous studies from this laboratory have demonstrated increased levels of expression of endogenous rat leukemia virus (RaLV) and 30S retrovirus-like sequences in liver and colon tumors induced by chemical carcinogens in rats. During the process of normal liver regeneration, the levels of RaLV RNA were dramatically increased, whereas the levels of 30S RNA did not change. The present study examined several factors that might influence the expression of these sequences in the Rat 6 embryo fibroblast cell line. Rat 6 cells in either log-phase or confluent cultures were treated with cycloheximide, the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), or the activated carcinogen benzo[a]pyrene diol epoxide (BPDE) for various periods of time up to 48 h. Northern blot analyses of total RNA indicated that cells in untreated cultures in log phase had higher levels of RaLV and 30S RNAs than did confluent cells. Within 10 h cycloheximide (2 or 10 micrograms/mL) markedly increased the levels of RaLV and 30S RNAs in both log-phase and confluent cells. BPDE (100 ng/mL) induced a marked increase in the levels of RaLV RNA at 4 to 10 h, which returned to the basal level by 24 h in the log-phase cells; but no significant change was seen in the confluent cells. The level of 30S RNA also increased moderately in the BPDE-treated log-phase cells and was maximal at 24 h; but no change was seen in confluent cells. Treatment with TPA (100 ng/mL) induced no significant increase in either RaLV or 30S RNA levels in the log-phase or confluent cells. The exposure of Rat 6 cells to 5-azacytidine (3 microM for 24 h) led to a marked increase in the levels of both RaLV and 30S RNAs, which persisted during at least 15 subsequent passages in the absence of the drug. Thus, inhibition of protein synthesis, DNA damage, or hypomethylation can increase the expression of certain endogenous retrovirus-like sequences, but an activator of protein kinase C does not.
Mol Carcinog 1990
PMID:Factors influencing the expression of endogenous retrovirus-like sequences in Rat 6 cells. 170 64

Recombinant DNA techniques have been used to develop Chinese hamster ovary cell lines for studying chemically induced genotoxicity. These cell lines express a specific cytochrome P450 isozyme responsible for the metabolism of polycyclic aromatic hydrocarbons and exhibit defined differences in DNA repair capacity. A bacterial gene (neo) conferring resistance to gentamicin was inserted into the pcD expression vector containing the mouse cytochrome P1450 (P450IA1) cDNA to facilitate the selection of transformed cells. This plasmid was introduced into the nucleotide-excision-repair-deficient UV5 cell line by electroporation. Transformed clonal isolates expressing the P1450 cDNA were identified by differential cytotoxicity assays using benzo[a]pyrene (B[a]P). One such clone, termed UV5P1, was mutagenized with ethyl methanesulfonate and selected for resistance to killing by UV radiation to derive a repair-competent clone that expresses similar P1450 activity to that of the parental cell line. Two repair-competent clones were selected and called 5P1R1 and 5P1R3. The resulting cell lines (UV5P1, 5P1R1, and 5P1R3) expressed arylhydrocarbon hydroxylase activity. UV5P1 and 5P1R3 were compared in terms of cytotoxicity and mutagenicity after exposure to B[a]P. Induced mutations were measured at the adenine phosphoribosyltransferase (aprt) and hypoxanthine guanine phosphoribosyltransferase (hprt) loci. Repair-deficient UV5P1 cells were killed by B[a]P at concentrations below 0.1 microM, while the repair-proficient 5P1R3 cells showed no toxicity up to 60 microM. Mutation induction at both loci was also much more efficient in UV5P1 cells. These new cell lines provide a more sensitive system that can be used in a battery of assays to evaluate the genotoxicity of chemicals requiring P450IA1 metabolic activation and to assess the role of DNA repair in modulating the biological effects of DNA damage.
Mol Carcinog 1991
PMID:Expression of mouse cytochrome P450IA1 cDNA in repair-deficient and repair-proficient CHO cells. 179 88

A short term, in vivo mutagenesis assay has been developed utilizing a lacl target gene contained within a lambda ZAP shuttle vector which has been incorporated into transgenic mice. Following chemical exposure, the target gene was recovered from mouse genomic DNA by mixing the DNA with in vitro lambda phage packaging extract. Mutations within the lacl target were identified by infecting host E. coli with the packaged phage and plating on indicator plates containing Xgal. Phage plaques with mutations in the lacl appeared blue, while intact phage were colorless. The ratio of blue plaques to colorless plaques is a measure of the mutagenicity of the compound. This system was used to obtain significant induction (up to 74-fold) over background levels for a variety of compounds, including N-ethyl-N-nitrosourea, benzo(a)pyrene (BaP), cyclophosphamide, and methylnitrosourea. Sequence analysis of selected mutant clones derived from this system was accomplished through the use of partial filamentous phage origins which allow rapid transfer of the target gene from phage to plasmid. Sequence analysis of spontaneous mutants derived from the mice primarily found of base substitutions, differing markedly from the previous data for spontaneous mutations in lacl derived from E. coli, where the preponderance of mutations were found at a single site, a repeated tetramer sequence. Upon sequence analysis of BaP derived base substitutions, only transversions were obtained, consistent with the known mechanism of BaP mutagenesis. Use of the well-characterized lacl gene in transgenic mice should allow for extrapolation of the extensive pool of in vitro data to whole animals, as well as provide insight into the tissue specific effects of mutagenic compounds.
Environ Mol Mutagen 1991
PMID:Analysis of spontaneous and induced mutations in transgenic mice using a lambda ZAP/lacI shuttle vector. 183 79


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