Partially transformed, anchorage-independent human diploid fibroblasts result from overexpression of the c-sis oncogene: mitogenic activity of an apparent monomeric platelet-derived growth factor 2 species.

A human c-sis cDNA in an expression vector was introduced into human diploid fibroblasts by transfection or electroporation. Fibroblast clones showing an aberrant, densely packed colony morphology were isolated and found to overexpress a 3.6-kilobase sis mRNA species and associated immunoprecipitable platelet-derived growth factor (PDGF) 2 proteins. Parallel analyses in cell clones of sis mRNA expression and colony formation in agar indicated that, above a threshold, a linear, positive correlation existed between sis overexpression and acquired anchorage independence. The sis-overexpressing cells formed transient, regressing tumor nodules when injected into nude mice, consistent with the finite life span which they retained. Protein products generated from the transfected c-sis construct in two overexpressing clones were immunoprecipitated with anti-human PDGF antibodies. One clone contained an apparent PDGF dimer of 21 kilodaltons; the second clone contained only an apparent PDGF monomer of 12 kilodaltons, which was shown to account for all of the mitogenic activity present in the cells, essentially all of which was concentrated in the membrane fraction. The results demonstrate a clear link between sis overexpression and acquisition of a partially transformed, anchorage-independent phenotype, and when combined with previous observations of sis overexpression in human tumors, clearly implicate sis overexpression as a genetic mechanism which contributes to human cell transformation.

Structures of class pi glutathione S-transferase from human placenta in complex with substrate, transition-state analogue and inhibitor.

BACKGROUND: Glutathione S-transferases (GSTs) are detoxification enzymes, found in all aerobic organisms, which catalyse the conjugation of glutathione with a wide range of hydrophobic electrophilic substrates, thereby protecting the cell from serious damage caused by electrophilic compounds. GSTs are classified into five distinct classes (alpha, mu, pi, sigma and theta) by their substrate specificity and primary structure. Human GSTs are of interest because tumour cells show increased levels of expression of single classes of GSTs, which leads to drug resistance. Structural differences between classes of GST can therefore be utilised to develop new anti-cancer drugs. Many mutational and structural studies have been carried out on the mu and alpha classes of GST to elucidate the reaction mechanism, whereas knowledge about the pi class is still limited. RESULTS: We have solved the structures of the pi class GST hP1-1 in complex with its substrate, glutathione, a transition-state complex, the Meisenheimer complex, and an inhibitor, S-(rho-bromobenzyl)-glutathione, and refined them to resolutions of 1.8 A, 2.0 A and 1.9 A, respectively. All ligand molecules are well-defined in the electron density. In all three structures, an additionally bound N-morpholino-ethansulfonic acid molecule from the buffer solution was found. CONCLUSIONS: In the structure of the GST-glutathione complex, two conserved water molecules are observed, one of which hydrogen bonds directly to the sulphur atom of glutathione and the other forms hydrogen bonds with residues around the glutathione-binding site. These water molecules are absent from the structure of the Meisenheimer complex bound to GST, implicating that deprotonation of the cysteine occurs during formation of the ternary complex which involves expulsion of the inner bound water molecule. The comparison of our structures with known mu class GST structures show differences in the location of the electrophile-binding site (H-site), explaining the different substrate specificities of the two classes. Fluorescence measurements are in agreement with the position of the N-morpholino-ethansulfonic acid, close to Trp28, identifying a possible ligandin-substrate binding site.

Human fibroblast chromatin states as effectors of the DNA-binding characteristics of benzo[a]pyrene anti-7,8-dihydrodiol 9,10-epoxide and two nonalkylating DNA-binding molecules.

Pure populations of mitotic or nonmitotic diploid human fibroblasts (greater than 98% pure) were exposed to [3H]benzo [a]pyrene (CAS: 50-32-8) anti-7,8-dihydrodiol 9,10-epoxide: r-7,t-8 dihydroxy-t-9, 10-oxy-7,8,9,10-tetrahydrobenzo [a]pyrene (or anti-diol-epoxide). In addition, metaphase chromosomes, interphase chromatin, or naked DNA was isolated from the pure cell populations and then titrated to saturation with anti-diol-epoxide, chromomycin A3, or 3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide (ethidium bromide). At saturation, anti-diol-epoxide had covalently modified 1.5% of the total deoxyguanosine residues in naked DNA, and this was reduced to 29 and 15% of this level in saturating the available anti-diol-epoxide-binding sites in chromosomes or chromatin, respectively. A similar hierarchy of accessible binding sites (naked DNA greater than chromosomes greater than chromatin) was also observed for the noncovalent interaction of chromomycin A3 or ethidium bromide with the human cell DNA. Deproteinization of the chromosome or chromatin preparations returned the level of drug binding to that seen with naked DNA. The results clarify the association between proteins and DNA in human chromatin and suggest how cell-cycle-dependent changes in DNA-associated proteins or higher-order changes in protein-DNA conformation can act to alter the access of molecules to DNA-binding sites.

Relationship between benzo(a)pyrene-induced DNA base modification and frequency of reverse mutations in mutant strains of Salmonella typhimurium.

Salmonella typhimurium cells (TA98 and TA100) were incubated with [3H]benzo(a)pyrene ([3H]BP) and induced rat liver microsomes. The BP-induced cytotoxicity and His+ reverse-mutation frequencies were determined, and bacterial DNA hydrolysates were chromatographed on Sephadex LH-20. Analysis indicated three principal DNA adducts formed from two diastereoisomeric BP diol-epoxides and a 9-hydroxy-benzo(a)pyrene metabolite. An 8.6-fold increase in TA100 cell concentration in the microsome incubation was paralleled by a 7.2-fold decrease in total adducts per cell and a 7.4-fold decrease in mutation frequency. Separate TA98 incubations were titrated with increasing concentrations of [3H](+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene ([3H]anti BP diol-epoxide), [3H](+/-)-7 beta, 8 alpha-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene [3H]syn BP diol-epoxide), or [3H]9-hydroxybenzo(a)pyrene. Linear, nonsaturated increases in DNA adduct levels were seen up to the highest observed concentrations of 4.00 microM BP diol-epoxide or 6.00 microM 9-hydroxybenzo(a)pyrene in both TA98 and TA100 cells. The increasing adduct levels were accompanied by linearly increasing mutation frequencies. At equivalent concentrations of the two DP diol-epoxides, an average of 8.2-fold more base substitution mutations (TA100) were seen than frameshift mutations (TA98). The results also indicate significant differences in absolute mutagenic efficiency (mutation frequency/unit modified DNA) between these three covalent DNA ligands (TA98, syn BP diolepoxide greater than 9-hydroxy-4,5-epoxy-benzo(a)pyrene greater than anti BP diol-epoxide; TA100, 9-hydroxy-4,5-epoxy-benzo(a)pyrene greater than syn BP diol-epoxide greater than anti BP diol-epoxide).

Transforming growth factor-alpha expression produces only morphological transformants of diploid human fibroblasts.

Transforming growth factor-alpha (TGF-alpha) is a potent mitogen for a variety of epithelial and mesenchymal cells and is commonly expressed in many human tumors and tumor cell lines. Frequently, this creates a potential autocrine circuit for growth stimulation in these cells; however, this is occurring in a background of other mutation-generated events. To determine the significance of the TGF-alpha circuit alone, we expressed the human TGF-alpha cDNA in a diploid human foreskin fibroblast strain, 7-25, under the control of the cytomegalovirus immediate early promoter-enhancer region and screened transfectants for TGF-alpha expression by Northern analysis and by immunoprecipitation. Partially processed forms (M(r) 24,000 and 20,000) of the recombinant TGF-alpha were observed in cell lysates and a M(r) 5500 fully processed form was secreted by the fibroblasts into the media. TGF-alpha-expressing clones showed an altered morphology and an increased saturation density (1.4- to 2.1-fold) but did not exhibit anchor-age-dependent growth in soft agarose or the ability to form tumors in nude mice. Additionally, expression of recombinant TGF-alpha did not extend the lifespan of these fibroblast clones. Scatchard analysis revealed approximately 10(5) epidermal growth factor (EGF) receptors on the surface of these human fibroblasts, indicating that the failure of TGF-alpha expression to strongly transform these cells is not due to low EGF receptor levels. These data suggest that cell type plays an important role in determining the transforming ability of TGF-alpha.

Mutation at separate gene loci in Salmonella typhimurium TA100 related to DNA nucleotide modification by stereoisomeric benzo(a)pyrene 7,8-diol-9,10-epoxides.

Suspensions of Salmonella typhimurium TA100 or TA1535 cells were exposed to pure enantiomeric forms or racemic mixtures of 3H-labeled benzo(a)pyrene anti- or syn-7,8-dihydrodiol-9,10-epoxide. Diol-epoxide-induced cytotoxicity and mutation frequencies at the hisG and gpt loci were determined. Hydrolysates of diol-epoxide-modified bacterial DNA were also examined by high-performance liquid chromatography and the primary structure and level of diol-epoxide-nucleoside adduct species were related to the observed frequencies of reverse mutations at the mutant hisG46 codon (histidine prototrophy) or forward mutations at the gpt locus (8-azaguanine resistance). Significant differences in mutagenic efficiency (i.e., mutation frequency per mol DNA adduct) were observed for the different enantiomeric diol-epoxides (-anti = +/- syn much greater than, + anti) and the mutagenic efficiencies were the same at both loci. The combined results of the mutation and adduct characterizations suggest that there are basic differences in the structural configuration of each adduct species which are recognized during errant DNA repair and as a result lead to base changes at a frequency which is relatable to the configuration of the original adduct lesion.

CBP/cycA, a CCAAT-binding protein necessary for adhesion-dependent cyclin A transcription, consists of NF-Y and a novel Mr 115,000 subunit.

Cells of most tissues, with the exception of hematopoietic cells, require adhesion to an appropriate surface to grow. Cyclin A is needed for cell cycle progression at the G1-S transition, and appearance of cyclin A mRNA and protein in late G1 has been shown to be dependent on adhesion-initiated signals in normal rat kidney fibroblasts. Previously, we have reported that the adhesion-dependent activation of cyclin A transcription in late G1 is mediated by CBP/cycA (CCAAT-binding protein for cyclin A gene), a novel CCAAT-binding protein. Specific binding of CBP/cycA, a Mr 30,000/40,000/115,000 heterotrimeric protein complex, to the CCAAT element of the cyclin A promoter was detectable in growing but not in G0-arrested or nonadherent normal rat kidney cells. Here, we demonstrate that the Mr 30,000/40,000 subunits of CBP/cycA are identical with NF-YA and NF-YB, the two subunits of NF-Y. In addition, we show that, aside from CBP/cycA, NF-Y itself also binds to the CCAAT element of the cyclin A promoter. But, whereas the binding of CBP/cycA is adhesion and cell cycle dependent and correlates with the expression of cyclin A in late G1 phase, NF-Y itself seems to bind in a cell cycle-independent manner.

Expression of a rat glutathione-S-transferase complementary DNA in rat mammary carcinoma cells: impact upon alkylator-induced toxicity.

The role of glutathione-S-transferase (GST) in alkylator drug resistance has been studied in MatB rat mammary carcinoma cells. A series of GST transfectant cell lines was established by using an expression vector containing the complementary DNA for the rat GST Yc gene under regulation of the SV40 early region promoter and the antibiotic resistance plasmid pSV2neo. Transfectant cell lines expressing up to 4-fold higher total GST activity than in the parental wild type cell line were identified. Southern blot analysis confirmed a DNA fragment corresponding in size to the transfected GST Yc complementary DNA. Wild type MatB cells contain very low levels of Yc protein, whereas the Yc+ clones showed greatly increased amounts of the Yc subunit. The effect of increased GST Yc activity on the sensitivity of the transfected clones to various cytotoxic agents was assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell survival assay. The clones expressing recombinant GST Yc were more resistant to melphalan (6- to 12-fold), mechlorethamine (10- to 16-fold), and chlorambucil (7- to 30-fold). In late passage populations of the GST Yc+ clones that had been grown over a period of 14 months under continuous selection in G418, GST activity was decreased and it was paralleled by a decrease in Yc protein. These late passage clones with diminished GST Yc content also demonstrate a partial reversion toward the wild type phenotype as determined by cytotoxicity assays using melphalan, mustargen, and chlorambucil. Interstrand DNA cross-links induced by mechlorethamine were significantly lower at 0, 2, and 20 h posttreatment in one of the GST Yc+ clones when compared to wild type MatB cells. These studies indicate that GST Yc overexpression can confer resistance to alkylating agents and that this correlates with inhibition of DNA cross-link formation.

N-myc oncogene enhances mitogenic responsiveness of diploid human fibroblasts to growth factors but fails to immortalize.

The N-myc gene is amplified in several types of human tumors. To assess the role of the N-myc gene in the transformation of normal human cells, we transfected an N-myc expression vector into diploid human fibroblasts. Transfected clones were isolated and found to express the N-myc gene at levels similar to those seen in a tumor cell line (neuroblastoma LA-N-1) which contains an amplified N-myc gene. The level of N-myc expression decreased as the N-myc clones senesced. Clones expressing N-myc had an increased saturation density and an altered morphology but did not have an extended lifespan. Under low serum conditions, neither the clones expressing N-myc nor the control cells showed anchorage-dependent growth. Clones expressing N-myc were compared to control cells to determine if different growth factors would affect the ability of cells to grow in soft agarose. Clones expressing N-myc and the control cells did not grow in soft agarose supplemented with epidermal growth factor (EGF) or transforming growth factor-alpha (TGF-alpha). However, compared to control cells, clones expressing N-myc grew in agarose 2.8- to 18-fold higher in response to basic fibroblast growth factor (bFGF) and 5.5- to 55-fold higher in response to platelet-derived growth factor B-chain homodimer (PDGF-BB).

Functional analysis of the SIS proximal element and its activating factors: regulated transcription of the c-SIS/PDGF-B gene in human erythroleukemia cells.

The SIS proximal element (SPE) is essential for the basal transcription of the c-sis/PDGF-B gene as well as the lineage-specific, activated transcription of this gene seen in megakaryocytes. In gel mobility shift analyses, the SPE element forms three gel-shift complexes; the t(op) and b(ottom) complexes were detected in nuclear extracts from both untreated and phorbol 12-myristate 13-acetate ('tetradecanoylphorbol acetate', TPA) treated K562 cells, whereas the m(iddle) complex was detected only in nuclear extracts from TPA-treated K562 cells. Site-directed mutagenesis of the SPE revealed a CCACCC motif that was essential for promoter activity as well as the formation of all three SPE gel-shift complexes. Nested-deletion analyses showed that the SPE was required for TPA-inducibility of c-sis/PDGF-B transcription. Antibody supershift analyses demonstrated that the t gel-shift complex contained both Sp1 and Sp3, and that the b complex contained only Sp3. In vitro transcription assays demonstrated that both Sp1 and Sp3 could support c-sis/PDGF-B transcription independent of each other in untreated K562 cells. However, overexpression of Sp1/Sp3 failed to significantly increase the c-sis/PDGF-B transcription in K562 cells.

Mammalian glutathione S-transferase: regulation of an enzyme system to achieve chemotherapeutic efficacy.

The glutathione S-transferases are a family of Phase II detoxication enzymes that catalyze the conjugation of glutathione to a large variety of electrophilic compounds. In the 1990s, there have been many advances regarding the function of these enzymes in protecting a cell from the toxic effects of these electrophiles. The complexity of this enzyme family has been realized and much work has been performed to identify the specific roles played by individual isozymes in resistance to a variety of agents. Likewise, the determination of the crystal structure of these enzymes has allowed the identification of specific amino acid residues that are involved in the catalysis of important reactions. The important role that these enzymes play in carcinogenesis and in drug resistance has warranted an attempt to bring together these different subfields of glutathione S-transferase biology to investigate possible ways that this system could be regulated in therapeutically useful ways. In this report, we have reviewed the recent advances and ways in which this knowledge could be utilized in the advancement of the treatment of cancer.

Mutational substitution of residues implicated by crystal structure in binding the substrate glutathione to human glutathione S-transferase pi.

Site-directed substitution mutations were introduced into a cDNA expression vector (pUC120 pi) that encoded a human glutathione S-transferase pi isozyme to non-conservatively replace four residues (Tyr7, Arg13, Gln62 and Asp96). Our earlier X-ray crystallographic analysis implicated these residues in binding and/or chemically activating the substrate glutathione. Each substitution mutation decreased the specific activity of the enzyme to less than 2% of the wild-type. Glutathione-binding was also reduced; however, the Tyr7----Phe mutant still retained 27% of the wild-type capacity to bind glutathione, underlining the primary role that this residue is likely to play in chemically activating the glutathione molecule during catalysis.

High-level production and purification of biologically active proteins from bacterial and mammalian cells using the tandem pGFLEX expression system.

Because of the complexities involved in the regulation of gene expression in Escherichia coli and mammalian cells, it is considered general practice to use different vectors for heterologous expression of recombinant proteins in these host systems. However, we have developed and report a shuttle vector system, pGFLEX, that provides high-level expression of recombinant glutathione S-transferase (GST) fusion proteins in E. coli and mammalian cells. pGFLEX contains the cytomegaloma virus (CMV) immediate-early promoter in tandem with the E. coli lacZpo system. The sequences involved in gene expression have been appropriately modified to enable high-level production of fusion proteins in either cell type. The pGFLEX expression system allows production of target proteins fused to either the N or C terminus of the GST pi protein and provides rapid purification of target proteins as either GST fusions or native proteins after cleavage with thrombin. The utility of this vector in identifying and purifying a component of a multi-protein complex is demonstrated with cyclin A. The pGFLEX expression system provides a singular and widely applicable tool for laboratory or industrial production of biologically active recombinant proteins in E. coli and mammalian cells.

A system utilizing Epstein-Barr virus-based expression vectors for the functional cloning of human fibroblast growth regulators.

The acquired ability of adherent mammalian cells to grow in suspension is closely linked to tumorigenic transformation. The anchorage-independence phenotype is likely to result from bypassing an adherence-responsive cell-cycle check-point at the G1/S boundary of the cell cycle. In order to identify genes that are part of or act upon the anchorage signal transduction pathway, we have developed a system which allows functional cloning of regulatory genes by expression of libraries of cDNA inserts either in the sense or antisense direction. The system is comprised of two components: (i) the library expression vectors, CMV-EL and C1E-EL, containing EBoriP for replication in EBN A-1-expressing cells, an expression cassette with a multiple cloning site suitable for directional insertion of cDNA libraries generated by standard protocols, and loxP sites which allow rapid manipulation of recovered vectors without the use of restriction enzymes and (ii) the EBNA-1-producing cell line, BB-5, a derivative of the immortalized, non-tumorigenic and anchorage-dependent human fibroblast cell line, MSU1.1. The growth characteristics of BB-5 cells did not differ from its parental cell line. BB-5 cells supported the episomal replication of CMV-EL and C1E-EL and allowed recovery of the vector from Hirt lysates of transfected BB-5 cells. BB-5 cells transformed to anchorage-independent growth by transfection with a mutant c-Ha-ras gene inserted into CMV-EL could be accurately and efficiently identified in a background of non-transfected BB5 cells by screening for anchorage-independent colonies with the aid of computer-assisted image analysis.

Polymorphic electrophile response elements in the mouse glutathione S-transferase GSTa1 gene that confer increased induction.

Induced transcription of a battery of stress response genes in mammals, including several phase I and phase II drug-metabolizing enzymes, is regulated by the electrophile responsive element (EpRE). Because previous directed mutagenesis of nucleotide motifs within the large, composite EpRE were shown to affect transcription factor binding and associated induced expression of dependent genes, we hypothesized that naturally-occurring variation or polymorphism in the EpRE sequence, if found, could affect the induced expression of important protective genes like glutathione S-transferases, and that this could be an important determinant of cancer risk in humans and other mammals. To determine whether this occurred in nature, 32 strains and species of inbred mice were screened to examine the EpRE sequence present in the mGSTa1 promoter. Two species, Mus caroli and Mus spretus, showed TGAC-->TGGC mutations in the tandem TGAC motif. Inducibility (15-fold) of the variant Mus spretus EpRE sequence in a reporter gene construct in HepG2 cells was significantly increased versus the wild-type EpRE sequence (8-fold). A comparison of mGSTa1-induced expression in the livers of Mus spretus, Mus caroli, and BALB/cJ mice showed the highest level of mGSTa1 mRNA in livers from the Mus spretus and Mus carolimice. This naturally-occurring polymorphism within the EpRE domain is the first mutation with an associated phenotype to be reported within a promoter regulatory element of a drug metabolizing gene.

Benzo[a]pyrene-diol-epoxide-induced anchorage-independence in diploid human fibroblasts. Analysis of cellular protooncogenes.

Treatment of diploid human fibroblasts with stereoisomeric benzo[alpha]pyrene anti and syn diol epoxides has been shown to induce anchorage-independent clones of cells with a dose dependence and frequency [(0.5-12) X 10(-4)] not significantly different from mutations at the hypoxanthine-guanine phosphoribosyltransferase locus [(1-8) X 10(-4)] in these cells. The majority of the anchorage-independent clones that were picked retained their mutagen-induced, anchorage-independent phenotype through at least 20 generations of expansion in monolayer culture. No variant cells showing extended life-span were detected among survivors in any of the mutagen treatment groups (less than 1.6 X 10(-7) frequency). Extensive analysis of a pool of 15 cellular protooncogenes (Ha-ras, Ki-ras, N-ras, mos, fos, fes, myc, abl, sis, myb, erbA, erbB, src, raf, N-myc), using Southern and northern blot analysis, was done to determine whether mutagen-induced rearrangement, amplification or overexpression of any of these genes was responsible for the mutagen-induced, anchorage-independent phenotype. We found no evidence that the genomic arrangement or expression level of any of these genes had been altered, thus indicating that an alternative form of mutation, or an alternative gene not included in this screening was responsible for the mutagen-induced, anchorage-independent phenotype.

Effect of estrogen-containing oral contraceptives on urinary corticosteroid sulfate excretion.

The excretion of corticosteroid sulfates and free cortisol in urine, and the total plasma cortisol, have been determined in 41 women receiving an estrogen-containing oral contraceptive and 53 age-matched female controls. The contraceptive steroids caused increases in the urinary corticosteroid sulfates and plasma cortisol similar to those observed in pregnancy; urinary free cortiscol was unchanged.

Characteristics of microsomal reduction of benzo[a]pyrene 4,5-oxide.

NADPH-reduction of benzo[a]pyrene 4,5-oxide (BP-4,5-oxide) to BP required four components from rat liver: cytochrome P-450, NADPH cytochrome P-450 reductase, phosphatidylcholine and a soluble, heat-sensitive factor which was present in 105 000 X g supernatant and was also released from microsomes by sonication. The requirement for this factor contrasts with recently reported results from Sugiura et al. (Cancer Res., 40 (1980) 2910). Oxide-reduction was 40 times faster under anaerobic conditions, but oxygen did not affect the stimulation factor. This stimulation was highest (X15) at low concentrations of microsomal protein (less than 0.1 mg/ml) and was almost absent at high concentrations of microsomal protein (greater than 1 mg/ml). Oxide-reduction activity was proportional to microsomal protein concentration in the presence of added 105 000 X g supernatant, but for microsomes alone (greater than 0.1 mg/ml) exhibited a parallel plot with an intercept at 0.08 mg/ml microsomal protein. Stimulation was highest at high concentrations of BP-4,5-oxide and a linear plot of V-1 vs. [BP-4,5-oxide]-1 was only obtained in the presence of 105 000 X g supernatant (Km = 3 microM, Vmax = 3.3 nmol/mg/min). Microsomal hydration of BP-4,5-oxide (inhibited in reductase assays) was unaffected by 105 000 X g supernatant, suggesting that stimulation of oxide-reduction did not derive from solubilization of BP-4,5-oxide. Stimulation was observed in the initial rate of reaction and was independent of incubation time. Inhibition of lipid peroxidation, removal of peroxides and deoxygenation were all excluded as explanations of the stimulatory effect.

Lack of covalent binding of peroxisome proliferators nafenopin and Wy-14 643 to DNA in vivo and in vitro.

[3H][2-methyl-2-p-(1,2,3,4-tetrahydro-naphthyl)phenoxy] propionic acid (nafenopin), a hepatocarcinogenic peroxisome proliferator, when administered p.o. to normal intact and partially hepatectomized male F344 rats did not show any significant binding to DNA and RNA, but bound to proteins. The in vitro incubation of [3H]nafenopin and [3H]4-chloro-[6-(2,3-xylidino)pyrimidinylthio]acetic acid (Wy-14643), another peroxisome proliferator, with hepatic microsomes and calf thymus DNA also showed no significant binding of these chemicals to DNA.

Benzo[a]pyrene diol-epoxides: different mutagenic efficiency in human and bacterial cells.

Monolayer cultures of diploid human fibroblasts and suspensions of S. typhimurium TA100 cells were treated with [3H]-labelled enantiomeric forms of benzo[a]pyrene anti and syn 7,8-dihydrodiol 9,10-epoxides. In both cell types, all of the enantiomers induced the formation of mutant 6-thioguanine (human) or 8-azaguanine-(bacterial)resistant cells. Diol-epoxide-modified nucleosides from human and from bacterial DNA hydrolysates were characterized by HPLC and showed essentially the same adduct species for human and bacterial cells treated with the same enantiomers. There were substantial differences, however, in the efficiency with which structurally-different adduct species were converted to mutant genotypes. In human cells, the mutagenic efficiency (mutation frequency/unit modified DNA) of the respective adduct species (+ anti much greater than -anti = +/- syn) at the hprt locus was exactly the opposite of that seen at a similar gene locus (gpt) in TA100 (-anti = +/- syn greater than + anti). The results suggest that the structural configuration of adducts in genomic DNA is important in determining whether a mutant genotype will result, and likewise, that there are differences in specificity between the human and bacterial systems which process these adduct lesions.