Mutational specificity of the anti 1,2-dihydrodiol 3,4-epoxide of 5-methylchrysene

An SV40-based pS189 shuttle vector, which contained a supF target gene and was replicated in human cells (Ad293), was used to determine the mutational specificity of anti 5-methylchrysene 1,2-dihydrodiol 3,4-epoxide, the active metabolite of the environmentally prevalent carcinogen 5-methylchrysene. The frequency of supF mutants containing point mutations increased with dose to approximately 40 times the spontaneous frequency. The induced mutations were not randomly distributed but occurred preferentially at mutagenic hotspots, which were not all identical to those reported by others for benzo[a]pyrene dihydrodiol epoxide, a metabolite with similar chemistry.     

Sequence specificity of aflatoxin B1-induced mutations in a plasmid replicated in xeroderma pigmentosum and DNA repair proficient human cells.

The mutagenic spectrum induced by aflatoxin-DNA lesions in DNA repair deficient and repair proficient human cells was investigated. The reactive metabolite aflatoxin B1-8,9-epoxide was synthesized and reacted in vitro with the shuttle vector plasmid pS189. Plasmids were transfected into human fibroblasts and allowed to replicate, and the recovered plasmids were screened in indicator bacteria for plasmid survival and mutations in the supF marker gene. Sequence data were obtained from 71 independently arising mutants recovered from DNA repair deficient xeroderma pigmentosum (XP) cells [XP12BE(SV40)] and 60 mutants recovered from a DNA repair proficient cell line (GM0637). Plasmid survival was lower and mutation frequency higher with the XP cells, and the mutation hotspots differed substantially for the 2 cell lines. Most mutations (> 90%) were base substitutions at G:C pairs, only about one-half of which were G:C-->T:A transversions, the expected predominant mutation. One-third of the mutations at GG sites and none of those at isolated Gs were G:C-->A:T transitions. Tandem base substitutions also occurred only at GG sites and were found only with XP cells. The location of mutation hotspots with either cell line did not correlate with the level of modification within the sequence as assessed by a DNA polymerase stop assay. These results suggest that the DNA repair deficiency associated with XP can influence not only the overall frequency of mutations but also the distribution of mutations within a gene. The finding of transition mutations exclusively at GG sites may be of predictive value in attempts to link dietary aflatoxin exposure to cancers associated with specific mutations in the c-ras oncogene and the p53 tumor suppressor gene.     

The p53 tumor suppressor protein reduces point mutation frequency of a shuttle vector modified by the chemical mutagens (+/-)7, 8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, aflatoxin B1 and meta-chloroperoxybenzoic acid.

p53 has been postulated to be the guardian of the genome. However, results supporting the prediction that point mutation frequencies are elevated in p53-deficient cells either have not been forthcoming or have been equivocal. To analyse the effect of p53 on point mutation frequency, we used the supF gene of the pYZ289 shuttle vector as a mutagenic target. pYZ289 was treated in vitro by ultraviolet irradiation, aflatoxin B1, (+/-)7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and meta-chloroperoxybenzoic acid and then transfected into p53-deficient cells with or without a p53 expression vector. p53 reduced the mutant frequency up to fivefold when pYZ289 was treated with aflatoxin B1, (+/-)7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene or meta-chloroperoxybenzoic acid but not when it was ultraviolet-irradiated. The p53-dependent mutation frequency reduction was higher at a higher level of premutational lesions for aflatoxin B1 and (+/-)7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and at a lower level of lesions for meta-chloroperoxybenzoic acid. This suggests that the chemical mutagens produce, in a dose-dependent fashion, two kinds of DNA damage, one subject to p53-dependent mutation frequency reduction and the other not. These results indicate that p53 can reduce the point mutation frequency in a shuttle vector treated by chemical mutagens and suggest that p53 can act as guardian of the genome for at least some kinds of point mutations.     

Preferential mutagenesis at G.C base pairs by the anti 3,4-dihydrodiol 1,2-epoxide of 7-methylbenz[a]anthracene

The racemic anti-dihydrodiol epoxide of 7-methylbenz[a]anthracene preferentially induced mutations at G.C base pairs in the pS189 shuttle vector. Mutations were not randomly distributed throughout the supF target gene, but were concentrated at five hotspots. The hotspots for this agent did not correspond exactly to those produced by any other dihydrodiol epoxide examined to date, indicating that dihydrodiol epoxide structure and reactivity play a major role in determining mutagenic hotspots.     

N-methyl-N-nitrosourea-induced mutations in a shuttle plasmid replicated in human cells

The supF gene of the recombinant shuttle plasmid pZ190 (modified pZ189) was used as a target to study the nature of mutations induced by N-methyl-N-nitrosourea (MNU) in human cells. Treatment of the intact plasmid with MNU followed by its replication in human lymphoblastoid cells led to extensive inactivation and no detectable mutations of the plasmid. However, exposure of the supF DNA fragment alone, followed by its ligation into the vector, caused a ten-fold increase in mutant frequency when replicated in O6-methylguanine-DNA methyltransferase-deficient cells (from 0.54 x 10(-3) to 5.8 x 10(-3)) and an 80-fold increase when replicated in cells containing normal levels of the enzyme (from 0.047 x 10(-3) to 3.8 x 10(-3)). About 45% of the mutant plasmid molecules recovered from human cells contained deletions and insertions. Sixty to 70% of the mutant molecules of wild-type size contained a single-base substitution. Most of these changes were of the G.C----A.T type, consistent with the hypothesis that O6-methylguanine is the primary mutagenic adduct induced by MNU. However, the distribution of mutation sites was highly nonrandom; more than half of all mutations were localized at the G.C position 123, and the rest were distributed in about a dozen sites. The high yield of mutations induced in the supF DNA in a host cell whose capacity for the removal of O6-methylguanine far exceeded the amount present in the supF suggests that the repair of damages in extrachromosomal DNA may be inefficient. This is supported by the observation that the yield of mutations in supF transfected into lymphoblastoid cells devoid of repair activity for O6-methylguanine was comparable to that observed with repair-proficient host cells. The present data, together with results of mutations induced in pZ189 by other agents, strongly suggest that one major determinant of mutational hot spots is the structure of the target DNA itself.     

Mutations induced by aminofluorene-DNA adducts during replication in human cells

To gain insight into the mechanisms by which carcinogens induce mutations in human cells, we treated a shuttle vector, pZ189, carrying the supF gene as the target for mutations with N-acetoxy-N-trifluoroacetyl-2-aminofluorene (N-AcO-TFA-AF). The plasmids were allowed to replicate in human cell line 293, and the progeny plasmids were examined for the frequency and kinds of mutations induced in supF, as well as their specific location in the sequence of the supF gene. The plasmids were reacted with N-AcO-TFA-AF so as to obtain the deacetylated adduct N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), the principal adduct formed in DNA when mammalian cells are exposed to reactive derivatives of 2-acetylaminofluorene (AAF), including N-acetoxy-2-acetylaminofluorene. The results showed there was a linear relationship between the number of dG-C8-AF adducts per plasmid and the frequency of supF mutants induced. DNA sequencing of 47 independent mutants obtained from doses of N-AcO-TFA-AF that increased the frequency of mutants 9-15 times the background frequency and three independent mutants from lower doses showed that 92% contained point mutations, i.e. changes affecting one, or two, or three nearby bases, and that all of these point mutations involved G.C base pairs. Ninety eight percent of the point mutations were base substitutions, predominantly G.C----T.A transversions. 46% of these mutations occurred at four out of the 85 bp in the target gene (hot spots). The most prominent mutation hot spot was also the most prominent hot spot for adduct formation as judged by the frequency of termination of in vitro polymerization by the Klenow fragment on N-AcO-TFA-AF-treated plasmids.     

人细胞色素p450 IA2基因的构建及其功能检测──该基因在啮齿类细胞中代谢

本文首先构建了含人类细胞色素ｐ４５０ＩＡ２ｃＤＮＡ的ｐＺｉｐ－ｐ４５０ＩＡ２的真核表达载体，并分别将其转染到Ｖ７９细胞和原代大鼠肝上皮细胞中．Ｎｏｒｔｈｃｒｎ杂交和免疫组化实验显示，Ｖ７９转染细胞中有人ｐ４５０ＩＡ２的ＲＮＡ和蛋白水平的表达。细胞毒实验和ＨＧＰＲＴ位点突变率检测结果证明，外源性ｐ４５０ＩＡ２基因可在Ｖ７９细胞中代谢活化黄曲霉毒素Ｂ１．转染有ｐＺｉｐ－ｐ４５０ＩＡ２质粒的原代大鼠肝细胞可代谢活化５ｎｇ／ｍｌ的黄曲霉毒素Ｂ１．本研究说明了人ｐ４５０ＩＡ２ｃＤＮＡ在ＭＭＬＶ的５′ＬＴＲ系统驱动下，可在体外啮齿类细胞中代谢活化黄曲霉毒素Ｂ１．它为ＡＦＢ１致癌机制的研究提供了一个新的、更为有效的途径。     

NAD(P)H:quinone oxidoreductase 1 reduces the mutagenicity of DNA caused by NADPH:P450 reductase-activated metabolites of benzo(a)pyrene quinones.

The role of microsomal NADPH:cytochrome P450 reductase (P450 reductase) and cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1 or DT-diaphorase) in the mutagenicity of benzo(a)pyrene-3,6-quinone (BP-3,6-Q) was studied using supF tRNA gene as the mutational target. pUB3 carrying the supF tRNA gene upon transformation into the Escherichia coli ES87 cells exhibited a spontaneous mutation frequency of 0.62 x 10(-6). Chemical modification of the pUB3 DNA with BP-3,6-Q caused a fourfold increase in the mutation frequency, compared with the spontaneous mutations. P450 reductase catalysed metabolic activation of BP-3,6-Q into reactive products (semiquinone and reactive oxygen species), which caused a further increase in the mutation frequency to eightfold over spontaneous mutations. Oxygen radical scavengers (SOD and catalase) blocked the P450 reductase-activated BP-3,6-Q-induced stimulation of mutations. This indicates that redox cycling of the semiquinone leading to the generation of reactive oxygen species (ROS) was directly responsible for the increased mutation frequency of P450 reductase-activated BP-3,6-Q. Analysis of the mutation spectra revealed that P450 reductase-activated BP-3,6-Q showed a significantly higher preference for frameshift mutations, particularly deletions, compared with the spontaneous mutations and the mutations generated by benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE). The single most frequently observed mutation by P450 reductase-activated quinone (semiquinone + ROS) was deletion of a single guanosine. Among the base substitutions, G:C --> T:A, G:C --> A:T and G:C --> C:G were also noticed. Interestingly, NQO1 competed with P450 reductase and specifically prevented the P450 reductase-activated BP-3,6-Q-induced mutations. However, BP-hydroquinone (BP-3,6-HQ) generated during the metabolic reduction of BP-3,6-Q catalysed by NQO1 caused specific mutations involving the deletion of a single cytosine from the DNA sequence 5''-CCCCC-3'' in supF tRNA gene at a significantly high frequency. A similar cytosine deletion was also observed with benzoquinone hydroquinone (HQ), indicating that the deletion of cytosine is associated with a hydroquinone class of compounds. These results suggest that: (1) quinones and P450 reductase-activated products of quinones (semiquinones and ROS) are mutagenic compounds; (2) the mutational spectra of quinones, semiquinones and hydroquinones differ from each other with respect to their mutational frequency and specificity; (3) NQO1 competes with P450 reductase and protects the cells from quinone mutagenicity; and (4) the NQO1 -metabolized quinones (hydroquinones), if not eliminated, cause specific mutations that are not observed with quinones and P450 reductase-activated quinones (semiquinones and ROS).     

Spectrum of cis-diamminedichloroplatinum(II)-induced mutations in a shuttle vector propagated in human cells

The supF gene of the shuttle vector pZ189 was used as a target for the study of mutations induced by cis-diamminedichloroplatinum(II) (cis-DDP). Normal human repair-proficient fibroblasts and cis-DDP repair-deficient xeroderma pigmentosum (XP) cells were used as host cells to study the effect of cis-DDP on the inhibition of shuttle vector replication and mutagenesis. Transfection of cis-DDP-treated pZ189 into normal and XP cell lines resulted in a marked increase in the mutation frequency and a decrease in the replication efficiency of the vector. However, these effects were much greater for the plasmid propagated in XP cells. Atomic absorption spectroscopy showed that six to eight Pt-DNA adducts per plasmid were necessary to inhibit plasmid replication by 50% in normal cells. In contrast, only one to two Pt-DNA adducts were necessary to inhibit replication of the plasmid by 50% in XP cells. Analysis of mutation sites demonstrated that cis-DDP treatment resulted primarily in single and double mutations separated by one base and limited to a few locations within the 85-bp mature tRNA. Propagation of the cis-DDP-treated vector in either normal or XP cells led to predominantly transversion mutations at AGA, AGG, and GAG sites and a cis-DDP-associated deletion of 174 bp. Although mutations occurred at target sites for cis-DDP adduct formation, there was no correlation between sites of mutation and the most frequent sites of adduct formation.     

OGG1 protein suppresses G:C-->T:A mutation in a shuttle vector containing 8-hydroxyguanine in human cells

8-Hydroxyguanine (8-OHG) is an oxidatively damaged mutagenic base which causes G:C-->T:A transversions in DNA. OGG1 was cloned as a human gene encoding a DNA glycosylase that specifically excises 8-OHG from DNA in vitro. However, it was not clear whether OGG1 protein suppresses G:C-->T:A transversions caused by 8-OHG in human cells in vivo. In the present study we have examined the ability of OGG1 protein to suppress G:C-->T:A transversions caused by 8-OHG in human cells by bacterial suppressor tRNA (supF) forward mutation assay using a shuttle vector DNA, pMY189. Introduction of a single 8-OHG residue at position 159 of the supF gene in plasmid pMY189 resulted in a 130-fold increase in mutation frequency compared with untreated plasmid pMY189 after replication in the NCI-H1299 human lung cancer cell line. G:C-->T:A transversions at position 159 were detected in >90% of the supF mutants from the 8-OHG-containing plasmid. The mutation frequency of the 8-OHG-containing plasmid was significantly reduced by overexpression of OGG1 protein in NCI-H1299 cells and, in particular, the occurrence of G:C-->T:A transversion at position 159 in the supF gene was suppressed. Furthermore, frequencies and spectra of mutations of the untreated pMY189 plasmid did not differ significantly with overexpression of OGG1 protein. These results indicate that OGG1 protein has the ability to suppress G:C-->T:A transversions caused by 8-OHG in human cells in vivo.     

A spectrum of mutations induced by crotonaldehyde in shuttle vector plasmids propagated in human cells

A spectrum of crotonaldehyde-induced mutations in the supF gene of the shuttle vector plasmid pMY189 replicated in human fibroblast cells was examined. Base sequence analysis of 104 plasmids with mutations in the supF gene revealed that the majority of the mutations were base substitutions (85%) and the rest were frameshifts (15%). A single base substitution was most frequently found (47%), while 25% had multiple base substitutions and interestingly 13% had tandem (adjacent two) base substitutions. Of the base substitution mutations, 50% were G:C-->T:A transversions and 23% were G:C-->A:T transitions. The mutations were not distributed randomly but were located at several hotspots, most of which were G:C base pairs in 5'-AAGG-3' (or 5'-CCTT-3') sequences. Production of propanodeoxyguanosine adducts may be related to such specificity in the mutation spectrum.      

Sequence-dependent mutations in a shuttle vector plasmid replicated in a mismatch repair deficient human cell line.

We utilized a shuttle vector plasmid (pLSC) to assess the role of DNA sequence and mismatch repair on mutagenesis in human cells. pLSC contains an interrupted 29 bp mononucleotide poly(G) run within a bacterial suppressor tRNA gene, which acts as a highly sensitive mutagenic target for detection of base substitution and frameshift mutations. The frequency of spontaneous mutations in pLSC was found to be similar after replication in either the hMSH6 (GT binding protein) mismatch repair-deficient MT1 line or its parental, mismatch repair-proficient line, TK6. However, the classes of plasmid mutations showed distinct differences in the two cell lines. Single base deletions comprised 48% of the mutations in the 56 independent pLSC plasmids sequenced from MT1 cells while these represented only 18% of the 40 independent pLSC mutants sequenced from the wild-type TK6 cells (P = 0.001). Virtually all the deletions included the mononucleotide run. In contrast, in pSP189, which contains the unmodified supF tRNA without the mononucleotide sequence, no single base deletions were observed for either cell line (P < 0.001). UV treatment of pLSC and pSP189 resulted in a 12-140-fold increase in mutations in TK6 and MT1 cells. These were predominately single base substitution mutations without a large increase in deletion mutations in the mononucleotide run in pLSC. These data indicate that a mononucleotide poly(G) run promotes single base deletion mutations. This effect is enhanced in a hMSH6 mismatch repair-deficient cell line and is independent of UV-induced mutagenesis.     

Mutational specificity of the syn 1,2-dihydrodiol 3,4-epoxide of 5-methylchrysene

The mutational specificity of the syn dihydrodiol epoxide of 5-methylchrysene in the supF gene of the pSP189 vector was examined. Transversion mutations at GC pairs predominated with G → T and G → C changes accounting for 42 and 21% of total base change mutations. The types of mutations found reflect the previously determined chemical preference of this reactive species for reaction with deoxyguanosine residues in DNA.     

Nickel (II) enhances benzo[a]pyrene diol epoxide-induced mutagenesis through inhibition of nucleotide excision repair in human cells: a possible mechanism for nickel (II)-induced carcinogenesis

Nickel (II), a ubiquitous environmental and industrial contaminant, is a well-known human carcinogen, particularly in human lung cancer. Although by itself it is a weak mutagen, nickel (II) is able to significantly enhance the genotoxicity of other mutagens and carcinogens, such as polycyclic aromatic hydrocarbons (PAHs) and ultraviolet light. Certain human populations, especially cigarette smokers, are frequently exposed to both nickel (II) and PAHs. To understand the interplay of nickel (II) and PAHs in mutagenesis and human carcinogenesis, we used a shuttle vector mutagenicity assay to examine the effect of nickel (II) on (+/-) anti-7beta, 8alpha-dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydroxybenzo[a]pyrene (BPDE)-induced mutagenesis in human cells. BPDE is an activated metabolite of benzo[a]pyrene (BP), a major carcinogen in cigarette smoke. The shuttle vector pSP189 modified with BPDE was transfected into human cells with and without nickel (II) exposure. We found that nickel (II) exposure significantly enhanced BPDE-induced mutation frequency, but did not change BPDE-induced mutational spectrum in the supF gene of pSP189 plasmids replicated in nucleotide excision repair (NER)-proficient human cells. However, the enhancing effect of nickel (II) on BPDE-induced mutation frequency was not observed in NER-deficient human XPA cells. We also found that nickel (II) exposure of human cells did not change the spontaneous mutation frequency of the supF gene in NER-proficient or NER-deficient human cells, indicating that nickel (II) did not affect the replication fidelity in human cells. Using a plasmid containing a luciferase reporter gene and a host cell reactivation assay, we have found that nickel (II) exposure greatly inhibited the repair of BPDE-DNA adducts in NER-proficient but not in NER-deficient cells. Together these results strongly suggest that nickel (II) can greatly enhance the mutagenicity and genotoxicity of PAHs by inhibiting the NER pathway in human cells, and this may constitute an important mechanism for nickel (II)-induced human carcinogenesis.     

Aflatoxin B1-induced DNA adduct formation and p53 mutations in CYP450- expressing human liver cell lines

Epidemiological evidence has been supporting a relationship between dietary aflatoxin B1 (AFB1) exposure, development of human primary hepatocellular carcinoma (HCC) and mutations in the p53 tumor suppressor gene. However, the correlation between the observed p53 mutations, the AFB1 DNA adducts and their activation pathways has not been elucidated. Development of relevant cellular in vitro models, taking into account species and tissue specificity, could significantly contribute to the knowledge of cytotoxicity and genotoxicity mechanisms of chemical procarcinogens, such as AFB1, in humans. For this purpose a non-tumorigenic SV40-immortalized human liver epithelial cell line (THLE cells) which retained most of the phase II enzymes, but had markedly reduced phase I activities was used for stable expression of the human CYP1A2, CYP2A6, CYP2B6 and CYP3A4 cDNA. The four genetically engineered cell lines (T5-1A2, T5-2A6, T5-2B6 and T5-3A4) produced high levels of the specific CYP450 proteins and showed comparable or higher catalytic activities related to the CYP450 expression when compared to human hepatocytes. The T5-1A2, T5-2A6, T5-2B6 and T5-3A4 cell lines exhibited a very high sensitivity to the cytotoxic effects of AFB1 and were approximately 125-, 2-, 2- and 15-fold, respectively, more sensitive than the control T5-neo cells, transfected with an expressing vector which does not contain CYP450 cDNA. In the CYP450-expressing cells, nanomolar doses of AFB1-induced DNA adduct formation including AFB1-N7-guanine, -pyrimidyl and -diol adducts. In addition, the T5-1A2 cells showed AFM1-DNA adducts. At similar levels of total DNA adducts, both the T5-1A2 and T5-3A4 cells showed, at codon 249 of the p53 gene, AGG to AGT transversions at a relative frequency of 15x10(-6). In contrast, only the T5-3A4 cells showed CCC to ACC transversion at codon 250 at a high frequency, whereas the second most frequent mutations found in the T5-1A2 cells were C to T transitions at the first and second position of the codon 250. No significant AFB1-induced p53 mutations could be detected in the T5-2A6 cells. Therefore, the differential expression of specific CYP450 genes in human hepatocytes can modulate the cytotoxicity, DNA adduct levels and frequency of p53 mutations produced by AFB1.      

Copper-dependent site-specific mutagenesis by benzoyl peroxide in the supF gene of the mutation reporter plasmid pS189.

Benzoyl peroxide (BzPO) enhances tumor promotion and malignant conversion in mouse epidermis. DNA damage may contribute to these processes. BzPO reacts with Cu(I) to produce the benzoyloxyl radical, which in turn causes strand breaks in plasmid DNA. In this study we investigated whether BzPO with or without Cu(I) caused promutagenic DNA damage in the supF gene of the mutation reporter plasmid pS189 replicating in human Ad293 cells. Exposure of pS189 in vitro to BzPO (0.1-1 mM) inhibited plasmid replication; however, addition of Cu(I) (0.1 mM) did not augment BzPO-induced plasmid toxicity. Exposure to BzPO with or without 0.1 mM Cu(I) was also associated with a concentration-dependent increase in mutation frequency, up to > 100-fold above the spontaneous mutation frequency. Supplemental Cu(I) was not required for mutagenesis; however, it both raised the maximal mutation frequency observed and lowered the threshold concentration of BzPO necessary to discern mutagenesis above the spontaneous background. Neither the hydroxyl radical scavengers mannitol or DMSO, the spin trap N-tert-butyl-alpha-phenylnitrone, nor reduced glutathione altered BzPO/Cu(I)-induced mutagenesis; however, mutagenesis was suppressed by the chelator EDTA. Twenty-four of 32 individual BzPO/Cu(I)-induced mutants characterized by sequencing contained point mutations; 22/25 point mutations occurred at G-C base pairs. There were five large deletions and four small deletions. Three additional BzPO-induced mutants contained four point mutations, all occurring at G-C base pairs. Two BzPO/Cu(I)-induced mutational clusters at d(pGGG)-d(pCCC) sites were observed. These data suggest that BzPO may interact with Cu(I) bound to G-C base pairs in DNA to produce site-specific promutagenic DNA damage.     

Kinds of mutations found when a shuttle vector containing adducts of 1,6-dinitropyrene replicates in human cells

To gain insight into the mechanisms by which carcinogens induce mutations in human cells, we have been comparing the frequency and spectrum of mutations induced when a shuttle vector, pS189, carrying covalently bound residues of structurally related carcinogens, replicates in human 293 cells. In the present study, we investigated the mutagenic effects of N-hydroxy-1-amino-6-nitropyrene, a partially reduced derivative of 1,6-dinitropyrene (1,6-DNP). The results were compared with what was found previously in the same assay with N-hydroxy-1-aminopyrene, the partially reduced derivative of 1-nitropyrene. The shuttle vector plasmids were exposed to tritiated 1-nitro-6-nitrosopyrene for 1 h in the presence of ascorbic acid, which served as a reducing agent to generate N-hydroxy-1-amino-6-nitropyrene. 32P-Postlabeling showed that only a single adduct was formed, i.e. N-(deoxyguanosin-8-yl)-1-amino-6-nitropyrene. There was a linear increase in the number of adducts per plasmid as a function of applied concentration and also in the frequency of supF mutants as a function of adducts per plasmid, reaching 58.8 x 10(-4) above a background of 0.8 x 10(-4). When the frequency of mutants induced when plasmids carrying residues of 1,6-DNP replicated in the human cells was compared with that induced by 1-NP residues, the former was 1.8 times more mutagenic than the latter. Both carcinogens induced mainly base substitutions, primarily G.C----T.A transversions; but 1,6-DNP adducts produced a significant fraction of -1 frameshifts, with most of these located in a unique run of five Gs in the gene. Polymerase termination reactions indicated that 1,6-DNP adducts were formed at every guanine, but not elsewhere in the gene. The 'hot spots' for adduct formation were not perfectly correlated with 'hot spots' for mutation induction. This indicates that the ultimate biological effect of the chemical depends not only on the number of adducts originally formed, but also on such processes as cellular DNA repair, which may remove such adducts from the plasmids before DNA replication occurs, as well as on the structure of the neighboring bases at the site of the adduct.     

Stable expression of human cytochrome P450IA2 cDNA in a human lymphoblastoid cell line: role of the enzyme in the metabolic activation of aflatoxin B1

A human lymphoblastoid cell line stably expressing a human cytochrome P450IA2 cDNA was developed. This recombinant cell line displayed P450IA2 protein and estradiol 2-hydroxylase activity, neither of which was detected in the parental cell line. The recombinant cell line was also approximately 1000-fold more sensitive to the cytotoxicity and mutagenicity of the carcinogenic mycotoxin aflatoxin B1 than was the parent cell line. The increase in mutagenicity was supported by a corresponding increase in the level of aflatoxin B1 binding to DNA in cells expressing P450IA2 relative to control cells.     

Hydroquinones cause specific mutations and lead to cellular transformation and in vivo tumorigenesis.

Benzo(a)pyrene and benzene are human carcinogens. The metabolic activation of these compounds into ultimate mutagenic and carcinogenic metabolites is prerequisite for their carcinogenic effects. In this report, the mutagenicity and carcinogenicity of hydroquinones of benzo(a)pyrene and benzene was investigated to address two important questions: (1) do hydroquinones contribute to benzo(a)pyrene and benzene carcinogenicity; and (2) how safe is it to increase the levels of NAD(P)H:quinone oxidoreductase 1 (NQO1), a key enzyme in the generation of hydroquinone. The supF tRNA of the plasmid pSP189 was used as the mutational target in a cell-free and Chinese hamster ovary (CHO) cell system to study hydroquinone mutagenicity. RNA and protein-free pSP189 DNA was incubated in a cell-free system with benzo(a)pyrene-3,6-quinone and purified NQO1 or with benzoquinone hydroquinone to generate adducted pSP189 DNA. The adducted pSP189 DNA was transfected in human embryonic kidney cells Ad293. In the CHO cell system, monolayer cultures of CHO cells and CHO cells overexpressing NQO1 or P450 reductase were transfected with pSP189 vector DNA, treated with benzo(a)pyrene-3,6-quinone. The adducted and replicated pSP189 DNA was rescued from transfected Ad293 (cell-free system) and CHO cells (CHO cell system), digested with the restriction enzyme Dpn1 to remove unreplicated DNA followed by transformation in Escherichia coli MBM7070. The mutant colonies [white/pale blue on 5-bromo-4-chloro-3-indolyl beta-D-galactoside/isopropyl beta-D-thiogalactoside (X-gal/IPTG) plates] were selected, regrown and analysed by DNA sequencing. Mutagenesis experiments demonstrated that hydroquinones cause sequence-specific frameshift mutations involving deletion of a single cytosine from the DNA sequence 5''-172-CCCCC176-3'' or a single guanosine from the complementary strand sequence 5''-GGGGG-3'' in the supF tRNA gene. This mutation was specific to the hydroquinones, as it was not observed with quinones and other components of the redox cycling (semiquinones and reactive oxygen species). Exposure of BALBc/3T3 cells to hydroquinones resulted in cellular transformation leading to the loss of contact inhibition and regulation of cell growth. The transformation efficiency of BALBc/3T3 cells exposed to hydroquinones was significantly increased by the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), indicating that hydroquinones are excellent initiators that require additional co-carcinogens or promoters to exert an effect. The hydroquinone + TPA as well as hydroquinone-transformed BALBc/3T3 cells, when injected s.c. in severe combined immunodeficient (SCID) mice, produced tumours at 100% frequency. These results establish that hydroquinones lead to mutagenicity and carcinogenicity.     

Ultraviolet mutagenesis in a plasmid vector replicated in lymphoid cells from patient with the melanoma-prone disorder dysplastic nevus syndrome

The hereditary dysplastic nevus syndrome (DNS) is an autosomal dominant disorder in which affected individuals have increased numbers of dysplastic (premalignant) nevi and a greater than 100-fold increased risk of developing cutaneous melanoma. Epstein-Barr virus-transformed lymphoblastoid cell lines from patients with hereditary DNS have been shown to be hypermutable to UV radiation (M.I.R. Perera et al., Cancer Res., 46: 1005-1009, 1986). To examine the mechanism involved in this UV hypermutability, we used a shuttle vector plasmid, pZ189, which carries a 160-base pair marker gene, supF, and can replicate in human cells. pZ189 was treated with UV radiation and transfected into DNS6BE, a lymphoblastoid cell line from a patient with hereditary DNS. Plasmid survival after UV was similar with the DNS6BE line and with a lymphoblastoid cell line from a normal donor. Plasmid mutation frequency was greater with the DNS line in accord with the DNS cellular hypermutability. Base sequence analysis was performed on 69 mutated plasmids recovered from the DNS line. There were significantly more plasmids with single base substitution mutations (P less than 0.01) in comparison to UV-treated plasmids passed through normal fibroblasts. pZ189 hypermutability and an increased frequency of single base substitutions was previously found with a cell line from a melanoma-prone xeroderma pigmentosum patient. These differences may be related to the increased melanoma susceptibility in both DNS and xeroderma pigmentosum.