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ERCC1 is a gene for repairing DNA damage whose function is related to carcinogenic-induced tumorigenesis and the effectiveness of platinum therapies. Circular RNAs (circRNAs) are products of posttranscriptional regulation with pleiotropic effects on the pathogenesis of lung cancer. We aim to identify that specific circRNAs derived from ERCC1 can regulate key biological processes involved in the development of lung cancer. We performed bioinformatics analysis, in vitro experiments, and analyzed clinical samples, to determine the biological features of a certain ERCC1-derived circRNA termed as hsa_circ_0051488 in benzo[a]pyrene diol epoxide-induced malignant transformed cell and lung cancer cell. The well-established model of transformed cells provided an ideal platform for analyzing the molecular characteristics of this circRNA in the malignant transformation of lung epithelial cell, which supports that hsa_circ_0051488 functions in the onset and growth of lung squamous cell carcinoma (LUSC). Further analysis indicates that the absence of hsa_circ_0051488 promoted the proliferation of cells with the malignant phenotype. Extensive experiments confirm that hsa_circ_0051488 is present in the cytoplasm and functioned as a competing endogenous RNA. In particular, hsa_circ_0051488 binds to mir-6717-5p, thereby modulating the expression of SATB2 gene, a lung cancer suppressor. Furthermore, our in silico experiments indicate that SATB2 can inhibit multiple tumor pathways and its expression positively correlated with the tumor suppressor gene CRMP1. These findings suggest a possible regulatory mechanism of hsa_circ_0051488 in LUSC, and that the newly discovered hsa_circ_0051488/miR-6717-5p/SATB2 axis may be a potential route for therapeutic intervention of LUSC., (© 2021 Wiley Periodicals LLC.)

Benzo(a)pyrene (BaP) is an endocrine-disrupting pollutant present in various aspects of daily life, and studies have demonstrated that BaP exerts reproductive toxicity. We previously showed that BaP damages endometrial morphology and decreases the number of implantation sites in early pregnant mice, but the mechanisms underlying these effects remain unclear. The endometrial function is crucial for implantation, which is associated with endometrial cell apoptosis. In this study, we focused on the effect of BaP on endometrial cell apoptosis and the role of WNT signaling during this process. Pregnant mice were gavaged with corn oil (control group) or 0.2 mg·kg -1 ·day -1 BaP (treatment group) from Days 1 to 6 of pregnancy. BaP impaired endometrial function by decreasing the expression of HOXA10 and BMP2, two markers of receptivity and decidualization. WNT5A and β-catenin were activated in the BaP group. BaP affected the expression of apoptosis-related proteins and inhibited the apoptosis of endometrial stromal cells. In vitro, human endometrial stromal cells (HESCs) were treated with different concentrations of BaP (dimethyl sulfoxide (DMSO); 5, 10 µM). WNT5A and β-catenin were also upregulated in the BaP treatment group. HESC apoptosis was restrained by BaP. Inhibiting WNT5A by SFRP5 partially restored the effect of BaP on apoptosis. In summary, these results suggested that BaP exposure during early pregnancy activates WNT5A/β-catenin signaling pathway, which inhibits the endometrial cell apoptosis and potentially destroys endometrial function., (© 2018 Wiley Periodicals, Inc.)

The X-linked Pig-a gene encodes an enzyme required for the biosynthesis of glycosyl phosphatidylinositol (GPI) anchors. Pig-a mutant cells fail to synthesize GPI and to express GPI-anchored protein markers (e.g., CD90) on their surface. Marker deficiency serves as a phenotypic indicator of Pig-a mutation in various in vivo assays. Here, we describe an in vitro Pig-a mutation assay in L5178YTk +/- mouse lymphoma cells, in which mutant-phenotype cells are measured by flow cytometry using a fluorescent anti-CD90 antibody. Increased frequencies of CD90-deficient mutants were detected in cells treated with benzo[a]pyrene (B[a]P), N-ethyl-N-nitrosourea (ENU), ethyl methanesulphonate, and 7,12-dimethylbenz[a]anthracene, with near maximum mutant frequencies measured eight days after treatment. The CD90 deficiency in mutant cells quantified by flow cytometry was shown to be due to loss of GPI anchors in a limiting-dilution cloning assay using proaerolysin selection. Individual CD90-deficient cells from cultures treated with ENU, B[a]P, and vehicle were sorted and clonally expanded for molecular analysis of their Pig-a gene. Pig-a mutations with agent-specific signatures were found in nearly all clones that developed from sorted CD90-deficient cells. These results indicate that a Pig-a mutation assay can be successfully conducted in L5178YTk +/- cells. The assay may be useful for mutagenicity screening of environmental agents as well as for testing hypotheses in vitro before committing to in vivo Pig-a assays. Environ. Mol. Mutagen. 59:4-17, 2018. Published 2017. This article is a US Government work and is in the public domain in the USA., (Published 2017. This article is a US Government work and is in the public domain in the USA.)

Rat CYP1A1 promoter-luciferase, transiently transfected wild-type and 4S PAH receptor (glycine N-methyl transferase, GNMT)-transformed Chinese hamster ovary (CHO) cells were exposed to benzo[a]pyrene and assayed for luciferase activity as an indicator of CYP1A1 promoter activity. CHO cells transformed with the rat 4S PAH receptor/GNMT expression vector had twice the induction level of luciferase activity with respect to wild-type CHO cells in concert with previously published reports that the 4S PAH receptor/GNMT mediates benzo[a]pyrene induction of CYP1A1 gene expression. Lysates of GNMT-transformed CHO cells and wild-type H4IIE rat hepatoma cells exposed to benzo[a]pyrene were immuno-precipitated with anti-GNMT antibodies, separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membrane for Western blot analysis with anti-aryl hydrocarbon receptor nuclear translocator (ARNT, HIF-1β) antibodies. Results of this analysis indicated that the 4S PAH receptor/GNMT forms a hetero-oligomer (dimer?) with ARNT/HIF-1β which dissociates in the presence of B[a]P. These observations further indicate the role of GNMT (which has been shown to be multifunctional) and B[a]P in the induction of CYP1A1 and also a potential role of GNMT in the modulation of hypoxia inducible factor-1 function with respect to the HIF-1β subunit (ARNT)., (Copyright © 2011 Wiley-Liss, Inc.)

Curcumin has great potential as a chemopreventive and chemotherapeutic agent; however, its effects on human papillomavirus (HPV)-associated molecular events are inadequately explored. This study examined the effects of curcumin on HPV-associated pathways involved in developing cervical cancer. We demonstrate for the first time that curcumin treatment suppresses cervical cancer cell growth in a three-dimensional raft culture system. Curcumin also inhibits tumorigenic characteristics as shown by decreases in both clonogenic potential and cell motility. Additionally, our findings show that curcumin treatment inhibits the transcription of HPV16 E6/E7 as early as 6 h posttreatment and restores the expression of tumor suppressor proteins p53, retinoblastoma protein, and PTPN13. While smoking is a recognized risk factor for cervical cancer, the molecular effects of smoke carcinogens on the expression of HPV E6/E7 oncogenes are not well known. We show for the first time that exposure to benzo[a]pyrene (BaP), a tobacco carcinogen, increases the expression of HPV E7 oncoprotein suggesting a molecular link between smoking and cervical cancer. Importantly, curcumin decreases the BaP induced increase in the expression of HPV E7 oncoprotein. The results of this study clearly demonstrate that curcumin alters HPV-associated molecular pathways in cervical cancer cells. These novel findings imply that curcumin may be an effective chemopreventive and therapeutic agent for cervical cancer prevention and treatment., (© 2010 Wiley-Liss, Inc.)

As the postgenomic era continues to unfold, a new wave of scientific investigation is upon us focusing on the application of genomic technologies to study the meanings encrypted on the DNA code and the responses of living organisms to changes in their environment. Recent functional genomics studies in this laboratory have focused on the role of the aryl hydrocarbon receptor, a ubiquitous transcription factor, in genetic programming during renal development. Also of interest is the application of genomics investigations to the study of chronic medical conditions associated with early life exposures to environmental contaminants. Molecular evidence is discussed in this review within the framework of human molecular medicine.

The MDM2 gene is overexpressed in several human tumors and its product may be processed into various isoforms. Recently, alternative splicing forms of MDM2 mRNA have been detected in various types of tumors. In this study, lung tissue from human non small cell lung cancers was examined for MDM2 mRNA splicing variants by nested RT-PCR. Of the 117 lung cancer tissue samples analyzed, a total of 31 (26.5%) had splice variants for the MDM2 gene, while 59 (50.4%) had undetectable levels of MDM2 transcript. Further analysis indicated that the predominant variant for 26 of the 31 samples with alternative MDM2 splicing products was MDM2-657, a splice variant lacking exons 3-11. Significant associations were found between the frequency of alternative splicing and the gender and smoking habits of the patients. Approximately 36% of male patients had alternative splicing of MDM2 compared with only 9.5% of female patients (P = 0.008); 44.2% of the smoker patients had alternative MDM2 splice forms versus 16.2% of nonsmokers (P = 0.003). Furthermore, most normal lung cell lines examined possessed only full-length MDM2 mRNA, while among several lung cancer cell lines, only H1355 and CaLu-1 cells lacked alternatively spliced MDM2 transcripts. When H1355 cells were treated in vitro with the cigarette smoke carcinogen benzo[a]pyrene (B[a]P) or the B[a]P metabolite benzo[a]pyrene diolepoxide (BPDE), three MDM2 splicing products were detected by nested RT-PCR. Finally, with the use of several specific inhibitors, we found that BPDE-induced MDM2 mRNA alternative splicing in H1355 cells may occur through the PI3K or MAPK pathway. Overall, our results suggest that carcinogens present in cigarette smoke increase the risk of alternative MDM2 splicing, which is highly associated with lung cancer., ((c) 2005 Wiley-Liss, Inc.)

The human diet contains a variety of compounds that exhibit chemopreventive effects towards an array of xenobiotics. In the present study, the antigenotoxic potential of selected dietary constituents including Diallyl sulfide (DAS), Indole-3-carbinol (I3C), Curcumin (CUR), and Black tea polyphenols (BTP) has been evaluated in the Salmonella typhimurium reverse mutation and mammalian in vivo cytogenetic assays. In addition, the anticlastogenic effect of the above dietary constituents was identified towards Benzo(a)pyrene (BaP) and cyclophosphamide- (CP) induced cytogenetic damage in mouse bone marrow cells. The induction of BaP and CP induced chromosomal aberrations, micronuclei formation, and sister chromatid exchanges (SCEs) were found to be inhibited in a dose-dependent manner by DAS, I3C, CUR, and BTP. Thus the study reveals the antimutagenic potential of these dietary compounds towards BaP- and CP-induced genotoxicity in microbial and mammalian test systems., (Copyright 2003 Wiley-Liss, Inc.)

The aim of this study was to investigate the chemoprotective effects of mustard sprouts on benzo(a)pyrene [B(a)P]-induced DNA damage in the single cell gel electrophoresis (SCGE)/Hep G2 assay. This model combines the advantages of the SCGE assay with that of human-derived cells that possess inducible phase I and phase II enzymes. Treatment of the cells with small amounts of mustard juice (0.1-1.25 microl/ml) and B(a)P reduced the genotoxic effect of the carcinogen in a dose-dependent manner. Contrary to the results with the juice, unexpected synergistic effects were observed with allyl isothiocyanate (AITC, 0.3 microM), a breakdown product of sinigrin, which is contained in black mustard and many other cruciferous vegetables. Although these concentrations of AITC did not cause DNA damage per se, pronounced dose-dependent DNA damage was seen with higher concentrations of AITC (>or= 25 microM). In parallel with the comet assays, also enzyme measurements were carried out which showed that exposure of the cells to mustard juice (2.0 microl/ml) causes a moderate induction of ethoxyresorufin-O-deethylase, and more pronounced (approximately 2-fold) increase of the activity of glutathione-S-transferase. In conclusion, our findings indicate that i) mustard juice is highly protective against B(a)P-induced DNA damage in human derived cells and ii) that induction of detoxifying enzymes may account for its chemoprotective properties. iii) Furthermore, our findings show that the effects of crude juice can not be explained by its allyl isothiocyanate contents., (Copyright 2003 Wiley-Liss, Inc.)

Rat small intestinal epithelial cells and human colon adenocarcinoma cells cultured on Matrigel expressed the differentiation specific enzyme, sucrase-isomaltase, as determined by indirect immunofluorescence. Rat small intestinal epithelial cells, rat colonocytes, and human colon adenocarcinoma cells developed an altered morphology when cultured on Matrigel and became apoptotic within 24-48 h. Benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin caused a 2- and 5-fold induction, respectively, of ethoxyresorufin-o-deethylase activity in rat small intestinal epithelial cells cultured on Matrigel. Benzo[a]pyrene- or 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced ethoxyresorufin-o-deethylase activity in rat small intestinal epithelial cells cultured on plastic was not detected. 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment caused a 14-fold induction of transfected, rat CYP1A1-promoter-luciferase activity in rat small intestinal epithelial cells cultured on Matrigel. Benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment induced ethoxyresorufin-o-deethylase activity by 6- and 1.6-fold, respectively in rat colonocytes cultured on Matrigel. Induction of ethoxyresorufin-o-deethylase activity was not observed in rat colonocytes cultured on plastic. CYP1A1-promoter-luciferase activity was induced 3-fold by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat colonocytes cultured on Matrigel. Induction of CYP1A1-promoter-luciferase activity in rat small intestinal epithelial cells or rat colonocytes cultured on plastic was not observed. Ethoxyresorufin-o-deethylase activity in human colon adenocarcinoma cells, cultured on either plastic or Matrigel, was induced 7-fold by benzo[a]pyrene. 2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced ethoxyresorufin-o-deethylase activity was 2-fold greater in human colon adenocarcinoma cells cultured on Matrigel compared to cells cultured on plastic. Extracellular matrix-mediated differentiation and apoptosis of intestinal cells provide in vitro systems for study of the regulation of CYP1A1 expression, carcinogen activation in the gut and mechanism(s) of apoptosis of colon cancer cells., (Copyright 2002 Wiley-Liss, Inc.)

In the absence of a causal relationship between the incidence of sporadic breast cancer and occurrence of mutations in breast cancer susceptibility genes, efforts directed to investigating the contribution of environmental xenobiotics in the etiology of sporadic mammary neoplasia are warranted. Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants, which have been shown to induce DNA damage and disrupt cell cycle progression. In this report we discuss published data pointing to PAHs as a risk factor in carcinogenesis, and present findings generated in our laboratory suggesting that the mammary tumorigenicity of PAHs may be attributable, at least in part, to disruption of BRCA-1 expression by reactive PAH-metabolites. We report that benzo[a]pyrene (B[a]P), selected as a prototype PAH, disrupts BRCA-1 transcription in estrogen receptor (ER)-positive but not ER-negative breast cancer cells. The reduced potential for BRCA-1 expression in B[a]P-treated cells coincides with disruption of cell cycle kinetics and accumulation of p53. These effects are counteracted by the AhR-antagonist alpha-naphthoflavone (ANF), and in breast cancer cells expressing mutant p53 or the E6 human papilloma virus protein. We suggest that exposure to PAHs may be a predisposing factor in the etiology of sporadic breast cancer by disrupting the expression of BRCA-1., (Copyright 2002 Wiley-Liss, Inc.)

The objective of this study was to investigate whether polycyclic aromatic hydrocarbons (PAHs) contribute to the etiology of sporadic breast cancer by altering the expression of BRCA-1. Acute exposure to the PAH benzo[a]pyrene (B[a]P) inhibited in a time- and dose-dependent fashion cell proliferation and levels of BRCA-1 mRNA and protein in estrogen receptor (ER)-positive breast MCF-7 and ovarian BG-1 cancer cells. Moreover, the acute exposure to B[a]P abrogated estrogen induction of BRCA-1 in MCF-7 cells. The loss of BRCA-1 expression was prevented by the aromatic hydrocarbon receptor (AhR) antagonist alpha-naphthoflavone, suggesting participation of the AhR pathway. BRCA-1 exon 1a transcripts were downregulated by B[a]P faster than exon 1b mRNA was. Long-term exposure to B[a]P (40 nM for 15 mo) lowered BRCA-1 mRNA levels in subclones of MCF-7 and BG-1 cells, whereas expression of BRCA-1 in these clones was reverted to normal levels by washing out of B[a]P. The mechanisms of BRCA-1 repression by B[a]P were further investigated by examining the effects of the halogenated aryl hydrocarbon 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and the B[a]P metabolite 7r, 8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). While TCDD did not influence basal BRCA-1 mRNA and protein levels at any of the doses (from 10 nM to 1 microM) tested in this study, treatment with 50 nM BPDE drastically reduced BRCA-1 mRNA levels, indicating that metabolism of B[a]P to BPDE may contribute to downregulation of BRCA-1. Conversely, ER-negative breast MDA-MB-231 and HBL-100 cancer cells were refractory to treatment with B[a]P or TCDD and expressed constant levels of BRCA-1 mRNA and protein. We conclude that B[a]P may be a risk factor in the etiology of sporadic breast cancer., (Copyright 1999 Wiley-Liss, Inc.)

Background: A recently developed laser scanning cytometry technique was applied to cytometric studies to detect rapidly stable chromosomal aberrations induced by a carcinogen in a Chinese hamster fibroblast cell line, CHO-WBLT., Methods: Individual chromosomes were collected from metaphase cells by a syringe technique and spread on slides. The DNA content of each chromosome stained with propidium iodide was measured with a laser scanning cytometer (LSC). A characteristic DNA histogram, designated as the "laser scanning karyotype (LSK)," was obtained from about 20,000 chromosomes of CHO-WBLT cells. Each chromosome was confirmed morphologically under the microscope by using a "re-location" system built into the LSC., Results: A total of 21 chromosomes, including marker chromosomes specific to the cell line, were assigned to 10 major peaks in the LSK, which was analogous to the karyotype demonstrated with the classical Q-banding technique. In contrast, clonal sublines isolated after exposure to the carcinogen benzo[a]pyrene showed LSKs different from those found in untreated control cells, and seven of 20 clones were found to be abnormal, with a small number of chromosomal translocations and/or deletions, which were confirmed by Q-banding., Conclusions: The laser scanning cytometry technique was employed to detect stable chromosomal aberrations in CHO-WBLT cells after treatment with benzo[a]pyrene. The results obtained with this technique were comparable to those obtained by Q-banding; therefore, this method may be useful for rapid primary screening to detect stable, abnormal karyotypes induced by environmental chemicals and/or radiation.

The evaluation of the relationship between the dose to DNA of a genotoxic carcinogen and in vivo somatic cell mutagenesis may provide important information on the mechanisms leading to induced mutational events. The induction of DNA adducts and DNA mutations in different tissues of lacZ plasmid-based transgenic mice has been investigated following a single intraperitoneal administration of the polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P). DNA adducts were measured by (32)P-postlabeling at times between 1 and 28 days following injection and DNA mutations in the lacZ reporter gene were quantified using a highly efficient immunomagnetic purification of the lacZ-containing pUR288 plasmid, followed by electrotransformation of circularized plasmids into a host-restriction negative E. coli C (DeltalacZ, galE (-)) host. The major DNA adduct formed by B[a]P, N (2)-(10beta-[7beta,8alpha, 9alpha-trihydroxy-7,8,9, 10-tetrahydrobenzo(a)pyrene]yl-deoxyguanosine, reached maximum levels between 5 and 7 days followed by a gradual decrease. The induced mutant frequency reached a maximum between 7 and 14 days, after the DNA adduct level in a particular tissue had reached its apparent maximum between 5 and 7 days. The mutant spectrum, defined as the percentage of no-change and size-change mutations in a particular tissue, changed from predominantly size-change mutations in untreated tissues to predominantly no-change mutations in tissues displaying the highest B[a]P-induced mutant frequencies. Contrary to the assumption that there are no factors that can eliminate mutations once they exist, it was observed that mutant frequencies in the organs studied declined to background levels, which was accompanied by a change in the mutant spectrum from predominantly no-change mutations to predominantly size-change mutations., (Copyright 1999 Wiley-Liss, Inc.)

The antimutagenic effect of ethanolic extract of propolis (EEP) and honeybee (Apis mellifera) venom, both collected in the State of São Paulo, Brazil, was assessed by the Salmonella/microsome assay upon direct- and indirect-acting mutagens. EEP had inhibitory effect (in an ascending order) on the mutagenicity power of daunomycin (TA102), benzo(a)pyrene (TA100), and aflatoxin B(1)(TA98) and the venom acted against the mutagenicity of 4-nitro-o-phenylenediamine (TA98) and daunomycin (TA102). Teratogenesis Carcinog. Mutagen. 19:403-413, 1999., (Copyright 1999 Wiley-Liss, Inc.)

To examine the ability of estrogens and anti-estrogens to induce cellular transformation and genetic effects, Syrian hamster embryo (SHE) cells were treated with estrogens, 17beta-estradiol (E2) or diethylstilbestrol (DES), or with anti-estrogens, tamoxifen (TAM), toremifene (TOR) or ICI 164,384. Treatment with each substance for 1-3 days suppressed cellular growth in a dose-dependent manner. Colony-forming efficiency (CFE) increased following treatment of cells with E2 or DES for 48 hr at 3 or 10 microM but decreased at 20 or 30 microM. In contrast, CFE was increased by treatment with TAM, TOR or ICI 164,348 over the concentration range examined (1-30 microM). Treatment with each chemical at 1-30 microM for 48 hr caused morphological transformation of SHE cells in a dose-related fashion. The highest frequency was exhibited in SHE cells treated with DES at 20 microM and was 2 times higher than that induced by treatment with benzo[alpha]pyrene (B[alpha]P) at 4 microM. Transformation frequencies induced by other substances (E2, TAM, TOR and ICI 164,348) did not exceed that induced by the B[alpha]P treatment. TOR showed a higher transforming ability over all concentrations examined when compared to the other anti-estrogens (TAM and ICI 164,348). No significant increases in the frequencies of chromosomal aberrations were observed in SHE cells that were treated with any of the chemicals. However, treatment of SHE cells with each chemical induced a dose-dependent increase of aneuploid cells in the near diploid range. Our results indicate that the ability of the estrogens and anti-estrogens to induce numerical chromosomal abnormality may be involved in their cell transformation activity and potential carcinogenicity.

In this study we tested the suitability of the human epithelial lung cell line BEAS-2B for in vitro studies of lung carcinogenesis. The human bronchial epithelial lung cell line BEAS-2B, immortalized with an SV-40/Ad-12 hybrid virus construct, was treated for 24 hours with five different concentrations of the lung carcinogen benzo(a)pyrene (B[a]P) to assess the relationship between DNA adduct levels, cell cycle distribution, micronuclei formation (MN), colony forming efficiency (CFE), and anchorage independent growth (AIG). There appeared to be a strong linear correlation between B[a]P concentration and DNA adduct formation, but no difference in cell cycle distribution was observed after incubation with various concentrations of B[a]P. In the incubation range of 4 to 100 nM B[a]P, the number of DNA adducts was linearly correlated with colony formation in AIG and with the number of cells within individual colonies but not the number of colonies in the CFE test. At higher B[a]P concentrations, the clonal expansion of cells in the CFE and the number of colonies in the AIG declined. Also, the number of micronuclei increased with the formation of DNA adducts. It is concluded that after 24 hours of incubation with 100 nM B[a]P, the formation of BPDE-DNA adducts in the human epithelial lung cells BEAS-2B results in maximal induction of cell transformation. Because of this correlation between DNA adduct formation and lung epithelial cell transformation, the BEAS-2B cells seem suitable for in vitro studies on lung carcinogens.

The administration of Colcemid for collecting mitotic figures in a carcinogenesis study, using benzo(a)pyrene (BaP), diminished the experimental differences between exposed and control mice. A dose-related increase in noncollected mitotic index (n-mitotic index) was seen in keratinocytes in the dorsal epidermis of mice which received four weekly treatments of BaP at 16, 32 and 64 micrograms in 50 microliters of acetone. In contrast, the number of mitotic figures collected for 4 hr by Colcemid block (c-mitotic index) was depressed at 16 micrograms, unchanged at 32 micrograms, and elevated at 64 micrograms of BaP. Weekly treatments with 4,8 or 16 micrograms BaP for 3-8 months induced an elevation in both n-mitotic and c-mitotic indices. The differences in results produced by the two methods of determining mitotic index depended upon dose and duration of treatment with BaP. The administration of Colcemid to acetone-treated mice increased the labeling index (number of labeled cells) and reduced the rate of DNA synthesis (low grain count per keratinocyte nucleus). After chronic application of BaP, Colcemid abrogated the increase in labeling index, but produced no additional effect on the number of grains per labeled keratinocyte. The modifying effect of Colcemid was greatest when administered during the peak of the tissue response to BaP. A number of significant changes in morphology of the skin associated with chronic exposure to BaP were attenuated by the use of Colcemid.

T47D human breast cancer cells were grown in 1 microM benzo[a]pyrene (BaP) for 3.5 months, and 2 BaP-resistant (BaPr) variant cell lines (CS and C10) were isolated. Decreased aryl hydrocarbon (Ah)-responsiveness in the CS and C1O BaPr cells was characterized by lower (80 to 900/o) induction of CYP1A1-dependent activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), lower levels of the nuclear Ah receptor complex and significantly decreased Ah receptor mRNA levels. Nuclear estrogen receptor (ER) binding and ER mRNA levels were similar in wild-type and mutant cell lines, whereas epidermal growth factor receptor mRNA levels were significantly decreased in the variant BaPr T47D cells. 17beta-Estradiol induced proliferation of both wild-type and BaPr T47D cells, and TCDD inhibited this response but did not down-regulate nuclear ER levels. The unique characteristics of the BaPr T47D variant cells will be used to further elucidate the mechanism of interaction between the ER and Ah receptor signalling pathways.

Vitamin A appears to exert a protective effect against certain cancers. Epithelial cancers, such as those of the skin, bladder, oropharynx and respiratory tract, have the strongest association with vitamin A. These same cancers are causally associated with exposure to carcinogens such as benzo(alpha)pyrene (BP), a product of combustion found in cigarette smoke and charbroiled meat. This study was designed to determine whether BP exposure affects tissue vitamin A nutriture. Female Sprague-Dawley rats were randomized to purified diets, sufficient or deficient in vitamin A, and with or without 200 mg BP/kg feed. Rats were killed after 4 or 6 weeks. Serum, liver and lungs were assessed for vitamin A levels; trachea, stomach, small intestine and bladder were examined for histologic change. Lack of dietary vitamin A resulted in a profound decrease in vitamin A in the serum, liver and lungs (p less than .005). No histologic changes were evident in any tissues examined. Serum vitamin A was not affected by dietary BP. In vitamin-A-sufficient rats, dietary BP caused a significant decline in hepatic and lung vitamin A. In rats fed vitamin-A-deficient diets, dietary BP had no effect on tissue vitamin A. We conclude that chronic exposure to the carcinogen BP leads to tissue depletion of vitamin A, despite a vitamin-A-sufficient diet. We postulate that BP impairs tissue repletion by metabolizing incoming vitamin A rather than in situ vitamin A, since BP had no effect on tissue vitamin A levels in rats fed a diet devoid of vitamin A. This BP-induced vitamin depletion may eventually have a deleterious effect on epithelial tissue health, and may help to explain the association between vitamin A and cigarette-smoke-related cancers.

In the rat, expression of the CYP1A1 gene is closely associated with arylhydrocarbon hydroxylase (AHH) enzyme activity. AHH is an inducile enzyme activity known to play an important role in the bioactivation of polycyclic aromatic hydrocarbons (PAHs) to mutagenic and carcinogenic metabolites. PAH-induced expression of the CYP1A1 gene appears to be regulated by several trans-acting factors, including the Ah receptor and the 4S PAH-binding protein. In this study, we used the PAH isomers benzo[a]pyrene (BaP) and benzo[e]pyrene (BeP) to further evaluate the role of the 4S PAH-binding protein in induction of the CYP1A1 gene in H4-II-E rat hepatoma cells. Although BaP is believed to bind to both the Ah receptor and the 4S protein, BeP has been reported to bind exclusively to the 4S protein. The results of the study presented here indicate that BaP and BeP induce the expression of the CYP1A1 gene, as measured by ethoxyresorufin O-deethylase (EROD) activity, in a concentration-dependent manner. However, BaP is about 25 times as potent as BeP in inducing EROD activity in these cells. Slot-blot analysis of total RNA isolated from these cells indicated that BeP, BaP, and 3-methylcholanthrene increased the level of CYP1A1 mRNA expression. Sucrose-gradient analysis of BeP binding activity indicated that BeP bound with high affinity to the 4S PAH-binding protein, but not to the Ah receptor. These results suggest that the 4S protein may play a role in the PAH-induced expression of the CYP1A1 gene in rat H4-II-E cells.

Primary cultures of mouse epidermal keratinocytes from SENCAR mice were treated with 7,12-dimethylbenz(a)anthracene (DMBA), benzo(a)pyrene [B(a)P], (+/-)7 beta-8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene [(+/-)anti-BPDE], and (+/-)7 beta, 8 alpha-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene [(+/-)syn-BPDE] to examine the relationship between DNA adduct formation and the induction of unscheduled DNA synthesis (UDS). DNA adducts were measured as pmol hydrocarbon bound per mg of DNA, and UDS was quantitated autoradiographically as net grains per nucleus. A good correlation was observed between the levels of UDS detected and the amount of DNA adducts present in the cell population when comparing similar compounds within the linear dose-response range of 0.005 micrograms/ml-0.25 micrograms/ml. A higher rate of UDS for a given level of DNA adducts was interpreted as an increased efficiency of DNA repair. In some cases, an increase in the efficiency of DNA excision repair correlated with lower tumor-initiating activity. For this family of PAH, the concentration below which UDS could no longer be detected was approximately 0.01 microgram/ml. However, DNA adducts were measurable at concentrations of 0.01 and 0.005 micrograms/ml. The limits of detection of the current UDS assay in the SENCAR MEK culture system occurred at hydrocarbon adduct levels of approximately 10 pmol/mg DNA, or approximately 1 adduct per 3 x 10(5) bases. Additionally, the UDS assay was unable to detect DNA repair induced by the weakly carcinogenic PAHs, dibenz(aj)anthracene and 7-methyl-dibenz(aj)anthracene. The UDS assay did detect DNA repair by the more strongly carcinogenic PAH, 6-methylcholanthrene. These results suggest that the present UDS assay with MEKs is a useful assay for the rapid screening of potential genotoxic agents. However, the limits of sensitivity are such that the current assay may be unable to detect a low level of DNA damage induced by some weakly genotoxic (carcinogenic) agents. In addition, while the limits of sensitivity determined in these experiments apply to the polycyclic aromatic hydrocarbon class, other classes of genotoxic compounds such as alkylating agents or crosslinking agents may exhibit different thresholds of detection.

Recently, a number of publications have suggested that bone marrow cytogenetics may be used to detect anticarcinogenic or antimutagenic activity. In this work, 0.75% 2,[3]-tert-butyl-4-hydroxyanisole (BHA), fed in the diet for 2 weeks, was tested for its ability to reduce the frequency of benzo(a)pyrene (BP)-induced SCE in mouse bone marrow. C57BL/6 male mice, were injected i.p. with BP at 0, 33, 67, and 100 mg/kg body weight. The mean SCE/chromosome +/- s.e.m. for animals on control diet was 0 mg/kg, 0.108 +/- 0.005; 33 mg/kg, 0.225 +/- 0.011; 67 mg/kg, 0.289 +/- 0.012; 100 mg/kg, 0.311 +/- 0.013. The mean SCE/chromosome +/- s.e.m. for animals on the 0.75% BHA diet was 0 mg/kg, 0.105 +/- 0.006; 33 mg/kg, 0.224 +/- 0.009; 67 mg/kg, 0.262 +/- 0.013; 100 mg/kg, 0.326 +/- 0.012. There are no significant differences between animals on the control and BHA diets. Excretion of BP in urine over a 72 hr time period was significantly increased in animals on the BHA diet, at both low and high doses. Water-soluble metabolites accounted for all of this increase. It appears that bone marrow is not a good model for the gastrointestinal tract, and that short-term assays for anticarcinogens or antimutagens are more likely to be predictive if they are done in the target organs.

Certain strains of mice vary in their enzyme inducibility by polycyclic hydrocarbons, i.e., the strain C57 shows high and the strain DBA shows low inducibility of aryl hydrocarbon hydroxylase (AHH) by benzo[a]pyrene (BaP). The effect of this genetically determined difference on the clastogenic response to BaP was studied in 11 day old embryos after transplacental treatment. The four possible crosses, C57 and DBA inter se, C57 X DBA and DBA X C57, were used to determine the influence of the genetic background on the aberration yields in the embryos. Constitutive and induced AHH levels were measured in liver, bone marrow, and placenta of the pregnant females and in their embryos. Enzyme inducibility was high in tissues of C57 dams and in their homozygous or heterozygous embryos. In contrast, induction of AHH activity was low in tissues of DBA females and their homozygous embryos. The high BaP-induced AHH activity found in heterozygous embryos of DBA dams is in agreement with the dominant mode of inheritance for high AHH inducibility. The cytogenetic results showed that the clastogenic response was lowest in homozygous C57 embryos and highest in hybrid embryos independent of the genetic constitution of the dams. Homozygous DBA embryos showed an intermediate aberration yield. The AHH inducibility by BaP did not correlate quantitatively with the induced aberration rates. However, the data suggest that BaP activation in embryonic tissue on day 11 of pregnancy is sufficient to account for the clastogenicity in the fetuses. It is concluded that the genetic endpoint chromosomal breakage is not only determined by the formation of active BaP metabolites but also by genetically controlled detoxification of BaP, repair process, and the rate of transformation of primary DNA lesions into true DNA discontinuities.

The metabolism of benzo(a)pyrene [B(a)P] to organic soluble and water soluble metabolites by transformable C3H10T1/2CL8 mouse embryo fibroblasts was studied as a function of time, B(a)P concentration, and cell density. The total formation of organic-soluble and water-soluble metabolites increased with incubation time from 4 to 48 h and with B(a)P concentration from 4 to 40 microM. As cell density increased, the metabolic rate decreased for organic-soluble and water-soluble products between 6,300 and 54,000 cells/cm2 probably due to decreases in B(a)P concentrations to values below saturation. Specific organic-soluble metabolites identified were B(a)P-pre-9,10-diols, B(a)P-9,10-diol, B(a)P-7,8-diol, B(a)P-3,6-quinone, B(a)P-3-phenol, and B(a)P-9-phenol. Water-soluble metabolites were subjected to enzymatic hydrolysis with beta-glucuronidase and aryl sulfatase to identify specific conjugated products. The sulfate conjugated metabolites identified were B(a)P-7,8-diol, B(a)P-pre-9,10-diols, B(a)P-9,10-diol, and B(a)P-3,6-quinone. The beta-glucuronic acid metabolites identified were B(a)P-pre-9,10-diols, B(a)P-3,6-quinone, and B(a)P-3-phenol. Patterns of metabolite formation rates are discussed as to their possible effect on morphological transformation rates in C3H10T1/2 cells with respect to incubation time and cell density.

Previous studies have shown that the onset of DNA synthesis in Balb/c 3T3 cells appears to be regulated by a labile protein. We have found that induction of thymidine kinase (TK) activity, after quiescent cells are stimulated by the addition of serum, is similarly regulated by a labile protein. Eight hours after serum stimulation, a 6-h pulse of cycloheximide (CHM) caused an excess delay of 2 h in TK induction. A similar delay also was found in the induction of thymidylate synthase (TS). In contrast, the benzo(a)pyrene transformed 3T3 cell line, BP-A31, which had previously been shown to have no excess delay for the onset of DNA synthesis also had no excess delay for the induction of TK activity after a pulse of CHM. The induction of TK was inhibited by actinomycin D and dichlororibofuranosylbenzimidizole (DRB) suggesting a requirement for new RNA synthesis. It did not appear to depend on DNA synthesis as it was not blocked by aphidicolin. In conclusion, the induction of TK activity appears to be regulated by the same labile cellular signal as the onset of DNA synthesis, and to depend on an increase in the level of TK mRNA in late G1 or early S phase.