DNA repair gene deficiency does not predispose human bronchial epithelial cells to benzo(a)pyrene-induced cell transformation

The development of cost and time-efficient in vitro assays to predict carcinogenicity of chemicals has become a very important direction for toxicological research. In this study, we generated a series of human bronchial epithelial (HBE) cells defect in DNA repair gene excision repair cross-completion 1 (ERCC1), excision repair cross-completion 2 (ERCC2), ataxia-telangiectasia mutated (ATM) and mutS homolog 2 (MSH2), respectively. The inhibition of gene expression was verified by detection of mRNA and protein levels of respective genes. The suppression of these DNA repair genes has no impact on cell proliferation or cell transformation. Although we found that the transgenic HBE cells were more sensitive in benzo(a)pyrene (BaP)-induced DNA damages measured by cytokinesis-block micronucleus (CBMN) assay and comet assay, we failed to observe enhanced effects on induction of cell transformation. HBE cells defect in DNA repair pathways did not exhibit malignantly transformed phenotype up to 20 weeks of BaP treatment, indicating that the deficiency of ERCC1, ERCC2, ATM, or MSH2 alone did not shorten the latency of cell transformation. In contrast, we found that HBE cells expressing H-Ras or c-Myc were transformed 8 or 12 weeks after BaP treatment. These findings demonstrate that silencing of a single DNA repair gene does not confer cells susceptible to chemical-induced cell transformation.     

Phosphorylation of p53 by Cdk5 contributes to benzo[a]pyrene-induced neuronal apoptosis

Benzo[a]pyrene (B[a]P) is a ubiquitous carcinogenic pollutant in the environment, however, the potential neurotoxic effects of B[a]P has not been elucidated clearly. In the present study, we explored the potential involvement of p53 phosphorylation by Cdk5 in B[a]P-induced neuronal apoptosis at both in vitro and in vivo settings. For in vitro studies, primary cortical neurons isolated from the brains of Sprague Dawley (SD) rat pup were exposed to 0, 10, 20, and 40 μM of B[a]P for 12, 24, or 48 h. For in vivo studies, SD rats were injected intraperitoneally with 0, 1.0, 2.5, and 6.25 mg/kg of B[a]P every other day for 1, 2, or 3 months. Our results demonstrated that exposure to B[a]P caused a dose- and a time-dependent increase in neuronal apoptotic ratio in both in vitro and in vivo studies. There was also a dose- and a time-dependent upregulation of p35, p25, Cdk5, and phosphorylated p53 at Ser15 after B[a]P exposure. In order to explore whether B[a]P-induced increased neuronal apoptosis was through Cdk5/p53 pathway, roscovitine, a specific Cdk5 inhibitor, was applied to pretreat neurons prior to B[a]P exposure. The results showed that pretreatment of neurons with roscovitine partially rescued cells from B[a]P-induced apoptosis, and alleviated B[a]P-induced upregulation of phosphorylated p53 at Ser15. Our results suggest that Cdk5/p53 signaling pathway may be involved in B[a]P-induced neuronal apoptosis, which will provide information to further elucidate the molecular mechanisms of B[a]P-induced neurotoxicity.     

Inhibitory effects of S-allylmercaptocysteine against benzo(a)pyrene-induced precancerous carcinogenesis in human lung cells

The anti-cancer effects of oil-soluble organosulfur compounds in garlic in the initiation phase of carcinogenesis are known. However, there are few experimental studies investigating S-allylmercaptocysteine (SAMC), a water-soluble derivative of garlic. This study investigated whether SAMC prevented the carcinogen benzo(a)pyrene (B(a)P) from inducing precancerous activity in human lung cells (A549 cell line). A549 cells were either pre-treated (PreTM) or concurrently treated (CoTM) with 1 μM B(a)P and either 10 or 50 μM SAMC. The 50 μM PreTM group inhibited B(a)P-induced cell proliferation by approximately 100%. The 50 μM SAMC PreTM and CoTM inhibited the B(a)P-induced G2/M phase shift by 100% and 97%, respectively. Furthermore, the PreTM and CoTM groups exhibited the potential to reduce the generation of reactive oxygen species (ROS) relative to the B(a)P group by at least 78%. The SAMC PreTM elevated superoxide dismutase (SOD) by approximately 100%. In this study, we revealed the mechanisms involved in SAMC inhibition of B(a)P-induced carcinogenesis, including suppression of cell proliferation, cell cycle regulation, attenuation of ROS formation, inhibition of DNA damage, increase of SOD activity and inhibition of nuclear factor-kappa B (NF-κB) activity. SAMC appears to be a novel therapeutic candidate for the prevention and treatment of B(a)P-induced human lung cancer.     

Anti-clastogenic effect of magnolol on benzo(a)pyrene-induced clastogenicity in mice.

It was previously reported that magnolol strongly inhibited the mutagenicity induced by the indirect mutagens [benzo(a)pyrene (B(a)P), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-aminodipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2), 2-aminoanthracene (2AA), and 7,12-dimethylbenz[a]anthracene (DMBA)] in Salmonella typhimurium TA98 and TA100 in the Ames test, and that the mechanism of this anti-mutagenic effect may involve the inhibition of the metabolic activation of indirect mutagen enzymes. In this study, the in vivo anti-clastogenic effect of magnolol against clastogenicity induced by B(a)P was evaluated using the micronucleus test in mice. Animals were treated with an oral administration of magnolol (1, 10, and 100 mg/kg) at -24, 0, 24, 48, 72, and 96 h before a single intraperitoneal injection of B(a)P. Peripheral blood specimens were prepared 48 h after administration of B(a)P, and analyzed by the acridine orange (AO) technique. The results indicated that magnolol inhibited clastogenicity induced by B(a)P at various administration times. In order to elucidate the mechanism behind this effect, we measured the activity of the detoxifying enzymes [UDP-glucuronosyltransferase (UGT) and glutathione-S-transferase (GST)] and antioxidative enzymes [superoxide dismutase (SOD) and catalase] in the liver when treated with an oral administration of magnolol at various administration times. Its effect on clastogenicity created by exposure to oxidative DNA damage-inducing X-ray irradiation was also evaluated using the micronucleus test in mice. Results showed that magnolol increased the activity of both UGT and SOD enzymes, and also inhibited the clastogenicity induced by X-ray irradiation. Magnolol had an anti-clastogenic effect on B(a)P in the micronucleus test as well as an anti-mutagenic effect on indirect mutagens in the Ames test. The anti-clastogenic effect of magnolol was also suggested by the increases in UGT and SOD enzyme activity, and by the attenuation of oxidative damage induced by X-ray irradiation.     

A mathematical approach to benzo[a]pyrene-induced hematotoxicity

Benzo[a]pyrene (BaP) has been reported to exert a differential effect on murine hematopoiesis that is mouse strain specific. Interpretation of these results based solely on experimental data is restricted and leaves important questions unanswered. Therefore, a mathematical model of murine hematopoiesis was applied in order to: (1) identify the targets of BaP, (2) quantify the damage to target cells and (3) based on these results, interpret differences in strain susceptibility. Model analysis of the hematopoietic response of D2 and BDF₁ mice to a daily oral administration of 125 mg/kg BaP showed that proliferating hematopoietic cells are the targets of BaP. Within this group it was found that: (a) erythropoietic cells were the most susceptible to BaP, (b) granulopoietic cells showed a susceptibility half that of erythropoietic cells and (c) the susceptibility of stem cells ranged between that of erythropoietic and granulopoietic cells. This damage pattern was the same for both strains, indicating that the difference between the strains was quantitative. As cell destruction rates were about 3-fold higher for D2 than BDF₁ mice, it was concluded that D2 mice were about three times as susceptible to BaP as BDF₁ mice. The study showed that the mathematical model, in addition to experimental methods, provided an efficient tool for the analysis of BaP hematotoxicity.     

New candidate proteins for benzo(a)pyrene-induced spatial learning and memory deficits

Spatial learning and memory (LM) is a property of central importance in the nervous system, yet many of the molecular mechanisms for benzo(a)pyrene[B(a)P]-induced LM deficits remain enshrouded in mystery. In this study, influence of exposure to B(a)P on LM deficits in adult male Sprague-Dawley rats was evaluated by Morris water maze. Then morphological changes in the ultramicrostructure of hippocampal neurons were observed by transmission electron microscopy. Furthermore, to better understand the molecular changes that occur in B(a)P induced LM deficits, antibody-based protein microarrays was used to analyze protein expression changes in rats submitted to sub-chronic oral gavage of B(a)P (2 mg/kg for 90 days). Results suggested that rats in the B(a)P-treated groups have significantly impaired Morris water maze performance when compared to controls. Meanwhile, the B(a)P-induced neuronal damage was also found in the hippocampus under transmission electron microscopy. Our results demonstrate that LM deficits associated protein expression signatures could be identified from tissue proteomes, as well as potential biomarkers such as retinoic acid receptor b (RARb), synaptotagmin iosfomrs 1 (Syt1) and Brain-derived neurotrophic factor (BDNF), previously not found. This study, therefore, identifies, for the first time, multiple novel proteins that are dysregulated by B(a)P, which both enhance our understanding of B(a)P induced LM deficits and represent targets of novel therapeutics.     

KIF11, a plus end-directed kinesin,as a key gene in benzo(a)pyrene-induced non-small cell lung cancer

Evidence indicates that Benzo(a)pyrenediol-epoxide (BPDE) can damage lung cells, resulting in carcinogenesis with complex mechanisms. We aimed to explore the genes and pathway variations in this process. First, the key gene was screened out and identified through data mining, and then, it was in turn validated by bioinformatics analysis and experimental methods. Consequently, 106 up-regulated and 260 down-regulated differentially expressed genes were yielded, which were enriched in various pathways, such as Cell cycle, and p53 signaling pathway. Then, KIF11 was identified as the key gene. Overexpression of KIF11 in lung cancer had a correlation with advanced pathological grade, advanced T stage, and presence of lymph node metastasis, which predicted poor prognosis. In summary, the present study revealed that KIF11 might be a key gene in the tumorigenesis of BPDE-related lung cancer, raising the possibility of KIF11 as a target for BPDE-induced lung cancer prevention and therapy.     

Cytoprotective effect of mangiferin on benzo(a)pyrene-induced lung carcinogenesis in swiss albino mice

Antioxidants are one of the key players in tumourigenesis, and several natural and synthetic antioxidants have been shown to have anticancer effects. In the present investigation, the efficacy of mangiferin on the antioxidant status of benzo(a)pyrene-induced lung carcinogenesis in Swiss albino mice was assessed. The animals were divided into five groups. The animals in groups I and V were normal control and mangiferin control, respectively. Groups II, III and IV were administered with benzo(a)pyrene (50 mg/kg body weight, orally) for 4 weeks (twice a week) to induced lung carcinogenesis. Starting 1 week prior to benzo(a)pyrene administration, group III animals were treated with mangiferin (100 mg/kg body weight) in the diet for 18 weeks; 12 weeks after benzo(a)pyrene administration, group III animals were treated with mangiferin that continued until the end of the experiment period (18 weeks). At the end of the experiment period, the reactive oxygen species, glutathione and the activities of antioxidant enzymes were assessed in both lung and liver tissues. The levels of glutathione, superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, vitamin E and vitamin C were decreased in group II animals. However, in the mangiferin + benzo(a)pyrene-treated groups III and IV, the levels of GSH and the activities of antioxidant enzymes in both lung and liver were improved when compared with benzo(a)pyrene-induced group II animals. In addition, the finding that mangiferin decreased reactive oxygen species levels and enhanced antioxidant status suggests that this polyphenol might also be of value in the prevention of benzo(a)pyrene-induced lung carcinogenesis.     

Aldehyde dehydrogenase 1A1 up-regulates stem cell markers in benzo[a]pyrene-induced malignant transformation of BEAS-2B cells

Recently, Aldehyde dehydrogenase 1A1 (ALDH1A1) has been proposed to be a common marker of cancer stem cells and can be induced by benzo[a]pyrene (B[a]P) exposure. However, the underlying mechanism of how ALDH1A1 contributes to B[a]P-induced carcinogenesis in human bronchial epithelial cells remains unclear. Here, we found that B[a]P up-regulated expression levels of stem cell markers (ABCG2, SOX2, c-Myc and Klf4), epithelial-mesenchymal transition (EMT) associated genes (SNAIL1, ZEB1, TWIST and β-CATENIN) and cancer-related long non-coding RNAs (lncRNAs; HOTAIR and MALAT-1) in malignant B[a]P-transformed human bronchial epithelial cells (BEAS-2B-T cells), and these up-regulations were dependent on increased expression of ALDH1A1. The inhibition of endogenous ALDH1A1 expression down-regulated expression levels of stem cell markers and reversed the malignant phenotype as well as reduced the chemoresistance of BEAS-2B-T cells. In contrast, the overexpression of ALDH1A1 in BEAS-2B cells increased the expression of stem cell markers, facilitated cell transformation, promoted migratory ability and enhanced the drug resistance of BEAS-2B cells. Overall, our data indicates that ALDH1A1 promotes a stemness phenotype and plays a critical role in the BEAS-2B cell malignant transformation induced by B[a]P.     

Detoxifying effects of optimal hyperoxia (40% oxygenation) exposure on benzo[a]pyrene-induced toxicity in human keratinocytes

ABSTRACT

Detoxifying effects of hyperoxia, which is widely used in clinical practice, were investigated using HaCat cells (human keratinocytes) treated with benzo[a]pyrene (B[a]P) as a model agent to induce adverse effects in the skin. It is well-established that B[a]P may produce toxicities including cancer, endocrine disruption, and phototoxicity involving DNA damage, free radical generation, and down regulation of nuclear factor erythroid 2-related factor 2 (Nrf2). It is well-known that Nrf2 is associated increase of antioxidant enzyme catalase (CAT) or detoxification enzyme glutathione S-transferase (GST) in HaCat cells treated with B[a]P under optimal condition of hyperoxia (40% oxygenation) conditions. To further examine the underlying basis of this phenomenon, factors affecting the expression of Nrf2 were determined. Nrf2 was upregulated accompanied by a rise in p38 MAPK, sequestosome-1 (also known as p62) and NF-κB. In contrast, Nrf2 was downregulated associated with an elevation in glycogen synthase kinase 3 beta (GSK-3β) and peroxisome proliferator-activated receptor alpha (PPARα). Hyperoxia was also found to diminish DNA damage and generation of free radicals initiated in B[a]P-treated cells which was attributed to an significant rise of Nrf2, leading to elevated antioxidant activities or detoxification proteins including heme oxygenase 1 (HO-1), superoxide dismutase (SOD), glutathione peroxidase-1/2 (GPX-1/2), CAT, GST and glutathione (GSH). In addition, factors related to skin aging were also altered by hyperoxia. Data suggest that optimal hyperoxia exposure of 40% oxygenation may reduce cellular toxicity induced by B[a]P in HaCat cells as evidenced by inhibition of DNA damage, free radical generation, and down-regulation of Nrf2.     

Fisetin, a novel flavonol attenuates benzo(a)pyrene-induced lung carcinogenesis in Swiss albino mice

Lung cancer is the foremost cause of cancer mortality and is a growing economic burden worldwide. Fisetin (3,7,3′,4′-tetrahydroxyflavone), a naturally occurring flavonoid is found in vegetables and fruits possesses anti-oxidative, anti-inflammatory and anti-proliferative effects in a wide variety of cancer. In the present study it is hypothesized that fisetin may provide chemopreventive as well as chemotherapeutic effects against experimental lung carcinogenesis. The present study was designed to investigate whether fisetin confers anti-cancer action against benzo(a)pyrene [B(a)P] induced lung carcinogenesis. Treatment with fisetin significantly reduced the degree of histological lesions, restored the levels of lipid peroxidation (LPO), enzymic and non-enzymic anti-oxidants in B(a)P-induced mice. Anti-proliferative efficacy of fisetin was assessed by immunohistochemical analysis of proliferating cell nuclear antigen (PCNA) in B(a)P induced mice showed increased PCNA expression which is restored upon fisetin administration. Together, our results depicts that fisetin can be used as chemopreventive agent against lung cancer.     

Gaseous nitrogen oxide repressed benzo[a]pyrene-induced human lung fibroblast cell apoptosis via inhibiting JNK1 signals

Benzo[a]pyrene (B[a]P) is present in environmental pollution and cigarette smoke. B[a]P has been shown to induce apoptosis in hepatoma cells, human B cells, human ectocervical cells, macrophages, and rat lungs. Nitrogen oxides (NOx) are the other important indoor and outdoor air pollutants. Many studies have indicated that NO gas causes lung tissue damage both by its oxidative properties and free radicals. In our previous study we demonstrated that NO gas induced proliferation of human lung fibroblast MRC-5 cells. In this study we showed that NO gas inhibits B[a]P-induced MRC-5 cells apoptosis by cell cycle analysis. Western blot data revealed that NO gas increased the expressions of anti-apoptosis proteins (Bcl-2 and Mcl-1) and decreased the expression of apoptosis proteins (Bax, t-Bid, cytochrome c, FasL, and caspases) after B[a]P treatment. We further clarified that B[a]P-induced MRC-5 cell apoptosis via JNK1/FasL and JNK1/p53 signals. In conclusion, NO gas inhibited B[a]P-induced MRC-5 cells apoptosis via inhibition of JNK1 apoptosis pathway and induction of Bcl-2 and Mcl-1 anti-apoptosis pathway.     

Effect of Sapthaparna (Alstonia scholaris Linn) in modulating the benzo(a)pyrene-induced forestomach carcinogenesis in mice

The chemopreventive effect of various doses of hydroalcoholic extract of Alstonia scholaris (ASE) was studied on the benzo(a)pyrene (BaP) induced forestomach carcinoma in female mice. The treatment of mice with different doses, i.e. 1, 2 and 4 mg/ml ASE in drinking water before, during and after the treatment with carcinogen, exhibited chemopreventive activity. The highest activity was observed for 4 mg/ml ASE, where the tumor incidence (93.33%) was reduced by 6.67%. Similarly, the tumor multiplicity reduced (61.29%) significantly (P<0.02) at 4 mg/ml in the pre-post-ASE treated group. However, the pre or post-treatment of mice with 4 mg/ml ASE did not show chemopreventive activity. These findings are corroborated by micronucleus assay, where treatment of mice with ASE before, during and after carcinogen treatment reduced the frequency of micronuclei (MN) in the splenocytes in a dose dependent manner. The MN frequency reached a nadir at 4 mg/ml ASE, the highest drug dose which showed maximum chemopreventive action. The ASE treatment not only reduced the frequency of splenocytes bearing one MN but also cells bearing multiple MN indicating the efficacy of ASE in inhibiting mutagenic changes induced by BaP. The pre or post-treatment of mice with 4 mg/ml ASE also significantly reduced the frequency of BaP-induced MN in the splenocytes of treated animals.     

Enhancement of benzo(a)pyrene-induced sister chromatid exchanges as a consequence of glutathione depletion in vivo

Pretreatment of Chinese hamsters with phorone (diisopropylidene acetone) decreased hepatic glutathione (GSH) content to about 20% of the control level after 2 hours. The GSH S-transferase activities were not affected. As a consequence of reduced detoxication by GSH the potency of 3,4-benzo(a)pyrene to induce sister chromatid exchanges in vivo in bone marrow cells was significantly enhanced. Chemically induced distinct alterations in metabolism of mutagens/carcinogens are proposed as experimental models for pharmacogenetic and toxigenetic studies.     

In vitro malignant transformation of human bronchial epithelial cells induced by benzo(a)pyrene

In this study, the human bronchial epithelial cells (16HBE) were treated five times with 10μM benzo(a)pyrene (BaP), followed by 20 passages culture, and the in vitro BaP-induced malignant transformation of 16HBE cells was established. Five colonies in soft agarose were then amplified and donated as T-16HBE-C1～5 cells, respectively. T-16HBE-C1～5 cells can form tumors subcutaneously in nude mice. Histopathological changes in the tumors indicated nests growth, high nuclear-cytoplasmic ratios, coarse and clumped chromatin, numerous and distinctly atypical mitoses, cell necrosis and surrounding normal adipose, muscle and connective tissue immersed. In addition, lung metastasis was observed in nude mice in T-16HBE-C1, 3 and 4 groups. In vitro cell migration assay results indicated that T-16HBE-C2～5 cells showed much lower migration capabilities than 16HBE cells. Western blotting analysis showed that the expressions of p53 and p-Akt (Ser473) in T-16HBE-C1～5 cells were significant higher than those in 16HBE cells. Our results demonstrated that BaP could induce the malignant transformation of 16HBE cells, and p53 and p-Akt (Ser473) might play crucial roles in BaP-induced carcinogenesis. The five monoclonal cell lines (T-16HBE-C1～5) with different migration capabilities could be used as research models for further understanding the mechanisms of BaP-induced carcinogenesis and cell migration.     

Influence of aroclor 1254 on benzo(a)pyrene-induced DNA breakage, oxidative DNA damage, and cytochrome P4501A activity in human hepatoma cell line

Both polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) are important environmental pollutants. They coexist widely in the environment at very low levels. Numerous studies indicated that aroclor1254 (one of PCBs mixture) is the inducer of cytochrome P450 1A enzyme acitivity. Benzo(a)pyrene (BaP) can cause a variety of toxicities in vitro, such as oxidative DNA damage and genotoxicity. In the present study, HepG2 cells were treated with either BaP (50 microM) or aroclor1254 at concentrations of 11.5 (low), 23.0 (medium), and 46.0 microM (high) alone, or pretreated the cells with aroclor1254 (11.5, 23.0, and 46.0 microM), followed by BaP (50 microM). It was found that 7-ethoxyresorufin-O-deetylase (EROD) activities of HepG2 cells exposed to either BaP or aroclor 1254 increased. DNA damage measured by DNA migration and the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) also increased in cells exposed to BaP, but not in cells exposed to aroclor1254. Under the Aroclor 1254 pretreatment condition, BaP-induced EROD activities was enhanced in cells exposed to the medium and high concentrations of aroclor1254 (P < 0.01 for both), whereas in all pretreatment groups aroclor1254 significantly increased BaP-induced DNA migration (P < 0.01 for all) and the 8-OHdG formation (P < 0.05 for all). In addition, there was positive correlation between the EROD induction activity and Olive tail moment (r(2) = 0.958, P < 0.01) or the levels of 8-OHdG (r(2) = 0.992, P < 0.01). The findings suggest that under the experimental conditions aroclor1254 may enhance BaP-induced DNA migration and oxidative DNA damage in HepG2, due to inducing CYP1A enzyme activity.     

Protective effects of lemongrass essential oil against benzo(a)pyrene-induced oxidative stress and DNA damage in human embryonic lung fibroblast cells

Benzo(a)pyrene (BaP) was a well-known environmental pollutant, numerous studies had implicated BaP as a causative agent in human cancer, particularly lung cancer. The lemongrass essential oil (LEO) possessed various pharmacological activities, especially the anti-oxidative stress and cancer prevention. In the current study, human embryonic lung fibroblast (HELF) cells were treated with 25?mM BaP in the absence or presence of 0.5%, 1% or 2.5% LEO and the cell viability and levels of oxidative stress (OS) and DNA damage in the cells were then measured. Nineteen chemical constituents were identified in LEO, with citral being the main component, representing about 68.78%. LEO was able to protect the HELF cells against BaP-induced loss in cell viability, achieving a maximum of 95.58% cell viability at the 0.5% concentration. Treatment of HELF cells with BaP alone significantly increased the level of Malondialdehyde (MDA) and decreased superoxide dismutase (SOD) and catalase (CAT). However, these effects were suppressed when the cells were also treated with LEO, leading to enhanced levels of SOD and CAT activities (2.9- and 2-fold, respectively, compared with BaP treatment only) and reduced the level of MDA in the cells (43% reduction in malondialdehyde level). At the same time, LEO also reduced the level of DNA damage, as shown by a reduced level of 8-hydroxy-deoxyguanosine (8-OHdG). Taken together, the results showed that LEO offered protection against BaP-induced OS and DNA damage, suggesting that LEO could be a promising agent for lung cancer chemoprevention.     

Diallyl trisulfide as an inhibitor of benzo(a)pyrene-induced precancerous carcinogenesis in MCF-10A cells

Diallyl trisulfide (DATS) is a garlic organosulfide that is toxic to cancer cells, however, little is known about its effect in the initiation phase of carcinogenesis. We sought to determine whether DATS could inhibit the carcinogen, benzo(a)pyrene (BaP), from inducing precancerous activity, in vitro. MCF-10A cells were either pre-treated (PreTx) or concurrently treated (CoTx) with 1 μM BaP, and 6 or 60 μM DATS for up to 24 h. The DATS 6 and 60 μM CoTx inhibited BaP-induced cell proliferation by an average of 71.1% and 120.8%, respectively, at 6 h. The 60 μM DATS pretreatment decreased BaP-induced G2/M cell cycle transition by 127%, and reduced the increase in cells in the S-phase by 42%; whereas 60 μM DATS CoTx induced a 177% increase in cells in G1. DATS effectively inhibited (P < 0.001) BaP-induced peroxide formation by at least 54%, which may have prevented the formation of BaP-induced DNA strand breaks. In this study, we reveal mechanisms involved in DATS inhibition of BaP-induced carcinogenesis, including inhibition of cell proliferation, regulation of cell cycle, attenuation of ROS formation, and inhibition of DNA damage. At the doses evaluated, DATS appears to be an effective attenuator of BaP-induced breast carcinogenesis, in vitro.