Comparing the effects of an exposure to a polycyclic aromatic hydrocarbon mixture versus individual polycyclic aromatic hydrocarbons during monocyte to macrophage differentiation: Mixture exposure results in altered immune metrics.

Polycyclic aromatic hydrocarbons (PAHs) are generated by the incomplete combustion of carbon. Exposures correlate with systemic immune dysfunction and overall immune suppression. Real-world exposures to PAHs are almost always encountered as mixtures; however, research overwhelmingly centers on isolated exposures to a single PAH, benzo[a]pyrene (B[a]P). Here, a human monocyte line (U937) was exposed to B[a]P, benz[a]anthracene (B[a]A), or a mixture of six PAHs (6-MIX) to assess the differential toxicity on monocytes. Further, monocytes were exposed to PAHs with and without CYP1A1 inhibitors during macrophage differentiation to delineate PAH exposure and PAH metabolism-driven alterations to the immune response. U937 monocytes exposed to B[a]P, B[a]A, or 6-MIX had higher levels of cellular health and growth not observed following equimolar exposures to other individual PAHs. PAH exposures during differentiation did not alter monocyte-derived macrophage (MDM) numbers; however, B[a]A and 6-MIX exposures significantly altered M1/M2 polarization in a CYP1A1-dependent manner. U937-MDM adherence was differentially suppressed by all three PAH treatments with 6-MIX exposed U937-MDM having significantly more adhesion than U937-MDM exposed to either individual PAH. Finally, 6-MIX exposures during differentiation reduced U937-MDM endocytic function significantly less than B[a]A exposed cells. Exposure to a unique PAH mixture during U937-MDM differentiation resulted in mixture-specific alterations of pro-inflammatory markers compared to individual PAH exposures. While subtle, these differences highlight the probability that using a model PAH, B[a]P, may not accurately reflect the effects of PAH mixture exposures. Therefore, future studies should include various PAH mixtures that encompass probable real-world PAH exposures for the endpoints under investigation.

PrimPol-dependent single-stranded gap formation mediates homologous recombination at bulky DNA adducts.

Stalled replication forks can be restarted and repaired by RAD51-mediated homologous recombination (HR), but HR can also perform post-replicative repair after bypass of the obstacle. Bulky DNA adducts are important replication-blocking lesions, but it is unknown whether they activate HR at stalled forks or behind ongoing forks. Using mainly BPDE-DNA adducts as model lesions, we show that HR induced by bulky adducts in mammalian cells predominantly occurs at post-replicative gaps formed by the DNA/RNA primase PrimPol. RAD51 recruitment under these conditions does not result from fork stalling, but rather occurs at gaps formed by PrimPol re-priming and resection by MRE11 and EXO1. In contrast, RAD51 loading at double-strand breaks does not require PrimPol. At bulky adducts, PrimPol promotes sister chromatid exchange and genetic recombination. Our data support that HR at bulky adducts in mammalian cells involves post-replicative gap repair and define a role for PrimPol in HR-mediated DNA damage tolerance.

Gut microbiota and undigested food constituents modify toxin composition and suppress the genotoxicity of a naturally occurring mixture of Alternaria toxins in vitro.

Molds of the genus Alternaria produce several mycotoxins, some of which may pose a threat for health due to their genotoxicity. Due to the lack of adequate toxicological and occurrence data, they are currently not regulated. Interactions between mycotoxins, gut microbiota and food constituents might occur after food ingestion, modifying the bioavailability and, therefore, overall toxicity of mycotoxins. The present work aimed to investigate the impact of in vitro short-term fecal incubation on the in vitro DNA-damaging effects exerted by 5 µg/mL of an Alternaria alternata extract, containing, among others, 15 nM alternariol, 12 nM alternariol monomethyl ether, 241 nM altertoxin II and 301 nM stemphyltoxin III, all of which are known as genotoxic. The involvement of microorganisms, undigested food constituents and soluble substances of human fecal samples in modifying the composition and the genotoxicity of the extract was investigated through the application of LC-MS/MS analysis and comet assays in HT-29 cells. Results showed that the potential of the mycotoxins to induce DNA strand breaks was almost completely quenched, even before anaerobic incubation, by contact with the different fractions of the fecal samples, while the potency to induce formamidopyrimidine DNA glycosylase (FPG)-sensitive sites was only slightly reduced. These effects were in line with a reduction of mycotoxin concentrations found in samples analyzed by LC-MS/MS. Although a direct correlation between the metabolic activity of the gut microbiota and modifications in mycotoxin contents was not clearly observed, adsorptive phenomena to bacterial cells and to undigested food constituents might explain the observed modifications.

Toxicity assessment of Fluoranthene, Benz(a)anthracene and its mixed pollution in soil: Studies at the molecular and animal levels.

An increasing amount of Fluoranthene (Fla) and Benz(a)anthracene (BaA) is being produced and used, eventually entering the soil sediments. The accumulation of Fla and BaA will cause poisoning to typical enzymes (α-Amylase) and organisms (Eisenia fetida) in soil. However, the studies about exploring and comparing the different effects of Fla, BaA and their joint effect at different levels are rarely reported. In this paper, the different effects of Fla, BaA and their mixed pollutant on α-Amylase were evaluated and compared at the molecular level, and the effect of Fla-BaA to the antioxidant system of earthworm (Eisenia fetida) was investigated from the aspects of concentration and exposure time at the animal level. The results showed that Fla-BaA had the greatest influence on the skeleton structure and the microenvironment of amino acid residue of α-Amylase compared to Fla and BaA, and in the mixed pollutant system, the joint effect mode was additive mode. The inhibitory effect of Fla-BaA on the activity of α-Amylase was also stronger than that of the system alone. The assays at the animal level showed that low concentrations (below 5 mg/kg) of Fla-BaA increased the activity of GSH-Px and SOD while high concentrations inhibited their activity. The POD that was activated throughout the experiment period suggested its key role in the earthworm antioxidant system. Changes in T-AOC and MDA showed that long-term and high-dose of Fla-BaA exposure inhibited the antioxidant capacity of Eisenia fetida, causing lipid peroxidation and damage to cells.

Genotoxicant exposure, activation of the aryl hydrocarbon receptor, and lipid peroxidation in cultured human alveolar type II A549 cells.

The aryl hydrocarbon receptor (AhR) transcription factor is activated by polycyclic aromatic hydrocarbons (PAH) and other ligands. Activated AhR binds to dioxin responsive elements (DRE) and initiates transcription of target genes, including the gene encoding prostaglandin endoperoxide synthase 2 (PTGS-2), which is also activated by the transcription factor NF-ĸB. PTGS-2 catalyzes the conversion of arachidonic acid (AA) into prostaglandins, thromboxanes or isoprostanes. 15-F2t-Isoprostane (IsoP), regarded as a universal marker of lipid peroxidation, is also induced by PAH exposure. We investigated the processes associated with lipid peroxidation in human alveolar basal epithelial cells (A549) exposed for 4 h or 24 h to model PAH (benzo[a]pyrene, BaP; 3-nitrobenzanthrone, 3-NBA) and organic extracts from ambient air particulate matter (EOM), collected in two seasons in a polluted locality. Both EOM induced the expression of CYP1A1 and CYP1B1; 24 h treatment significantly reduced PTGS-2 expression. IsoP levels decreased after both exposure periods, while the concentration of AA was not affected. The effects induced by BaP were similar to EOM except for increased IsoP levels after 4 h exposure and elevated AA concentration after 24 h treatment. In contrast, 3-NBA treatment did not induce CYP expression, had a weak effect on PTGS-2 expression, and, similar to BaP, induced IsoP levels after 4 h exposure and AA levels after 24 h treatment. All tested compounds induced the activity of NF-ĸB after the longer exposure period. In summary, our data suggest that EOM, and partly BaP, reduce lipid peroxidation by a mechanism that involves AhR-dependent inhibition of PTGS-2 expression. The effect of 3-NBA on IsoP levels is probably mediated by a different mechanism independent of AhR activation.

Nuclear factor of activated T cells as a marker of in vivo low-dose dibenzo[a,h]anthracene exposure.

We previously demonstrated that particulate matter ≤2.5 µm (PM2.5) suppresses the immune response in the spleen in vivo. Although PM2.5 includes the polycyclic aromatic hydrocarbon (PAH) such as dibenzo[a,h]anthracene (DBA), it is unclear whether PAH has a direct effect on the responses of splenocytes. In our study, the concentration of DBA used was approximately 0.8 µm, which is much lower than concentrations used in other toxicological studies of DBA. Although exposure to high concentrations of DBA is implicated in carcinogenesis, the effects of low doses of DBA on immune cells in vivo remain unclear. Here, we investigated the effects of low DBA doses on mouse splenocytes in vivo. Mice were administered dimethyl sulfoxide or DBA (0.4 or 0.8 µm) intratracheally. Twenty-four hours after treatment, the mice were killed and their splenocytes were collected. DBA treatment enhanced mitogen-induced cell proliferation and cytokine production in the mouse splenocytes. Furthermore, DBA enhanced splenic CD4+ and CD8+ cell proliferation and cytokine production. The nuclear factor of activated T cells (NFAT) was activated in CD4+ cells. DBA also activated nuclear factor-kappa B and CCAAT enhancer-binding protein pathways in CD11b+ cells. DBA-enhanced splenocyte activation was Toll-like receptor 2-, 4-, 9- and MyD88-independent. These results suggest that NFAT represents a promising marker for evaluation of the effects of DBA on T cells and T-cell-dependent antibody responses.

DNA reactivity of altertoxin II: Identification of two covalent guanine adducts formed under cell-free conditions.

Fungi of the genus Alternaria infest many agricultural crops and produce numerous mycotoxins, of which altertoxin II (ATX II) is one of the most mutagenic metabolites. ATX II carries an epoxide group but the formation of DNA adducts has not been demonstrated to date. We report now that ATX II gives rise to two covalent adducts with guanine when incubated with DNA under cell-free conditions. These adducts were demonstrated by LC-high resolution MS after enzymatic degradation of the incubated DNA to deoxynucleosides. The major adduct results from the covalent binding of ATX II, presumably through the epoxide group, to guanine, whereas the minor guanine adduct is derived from the major one by the elimination of two equivalents of water. In addition, a third adduct was detected, formed through covalent binding of ATX II to cytosine followed by the loss of two equivalents of water. The direct DNA reactivity of ATX II may explain its high mutagenicity.

Arsenite and cadmium promote the development of mammary tumors.

Previous studies demonstrate that the heavy metal cadmium and the metalloid arsenite activate estrogen receptor-alpha in breast cancer cells by forming a high-affinity complex with the ligand-binding domain of the receptor and that environmentally relevant doses of cadmium have estrogen-like activity in vivo. The present study showed that in estrogen-receptor positive cells, arsenite and cadmium increased the global expression of estrogen-responsive genes and that an environmentally relevant dose of arsenite also had estrogen-like activity in vivo. Similar to estrogens, exposure of ovariectomized animals to arsenite induced the expression of the progesterone receptor, GREB1, and c-fos in the mammary gland and the expression of complement C3, c-fos, and cyclin D1 in the uterus and the increase was blocked by the antiestrogen ICI-182,780. When virgin female animals were fed a diet, that mimics exposure to either arsenite or cadmium, and challenged with the chemical carcinogen dimethylbenzanthracene, there was an increase in the incidence of mammary tumors and a decrease in the time to tumor onset, but no difference in the total number of tumors, tumor multiplicity, or total tumor volume. Together with published results, these data showed that environmentally relevant amounts of arsenite and cadmium had estrogen-like activity in vivo and promoted mammary tumorigenesis.

The role of metabolism in the developmental toxicity of polycyclic aromatic hydrocarbon-containing extracts of petroleum substances.

In vitro assays presently used for prenatal developmental toxicity (PDT) testing only assess the embryotoxic potential of parent substances and not that of potentially embryotoxic metabolites. Here we combined a biotransformation system, using hamster liver microsomes, with the ES-D3 cell differentiation assay of the embryonic stem cell test (EST) to compare the in vitro PDT potency of two 5-ring polycyclic aromatic hydrocarbons (PAHs), benzo[a]pyrene (BaP) and dibenz[a,h]anthracene (DBA), and dimethyl sulfoxide extracts from five PAH-containing petroleum substances (PS) and a gas-to-liquid base oil (GTLb), with and without bioactivation. In the absence of bioactivation, DBA, but not BaP, inhibited the differentiation of ES-D3 cells into beating cardiomyocytes in a concentration-dependent manner. Upon bioactivation, BaP induced in vitro PDT, while its major metabolite 3-hydroxybenzo[a]pyrene was shown to be active in the EST as well. This means BaP needs biotransformation to exert its embryotoxic effects. GTLb extracts tested negative in the EST, with and without bioactivation. The PS-induced PDT in the EST was not substantially changed following bioactivation, implying that metabolism may not play a crucial role for the PS extracts under study to exert the in vitro PDT effects. Altogether, these results indicate that although some PAH require bioactivation to induce PDT, some do not and this latter appears to hold for the (majority of) the PS constituents responsible for the in vitro PDT of these complex substances.

Induction of the Antioxidant Response by the Transcription Factor NRF2 Increases Bioactivation of the Mutagenic Air Pollutant 3-Nitrobenzanthrone in Human Lung Cells.

3-Nitrobenzanthrone (3-NBA) is a suspected human carcinogen present in diesel exhaust. It requires metabolic activation via nitroreduction in order to form DNA adducts and promote mutagenesis. We have determined that human aldo-keto reductases (AKR1C1-1C3) and NAD(P)H:quinone oxidoreductase 1 (NQO1) contribute equally to the nitroreduction of 3-NBA in lung epithelial cell lines and collectively represent 50% of the nitroreductase activity. The genes encoding these enzymes are induced by the transcription factor NF-E2 p45-related factor 2 (NRF2), which raises the possibility that NRF2 activation exacerbates 3-NBA toxification. Since A549 cells possess constitutively active NRF2, we examined the effect of heterozygous (NRF2-Het) and homozygous NRF2 knockout (NRF2-KO) by CRISPR-Cas9 gene editing on the activation of 3-NBA. To evaluate whether NRF2-mediated gene induction increases 3-NBA activation, we examined the effects of NRF2 activators in immortalized human bronchial epithelial cells (HBEC3-KT). Changes in AKR1C1-1C3 and NQO1 expression by NRF2 knockout or use of NRF2 activators were confirmed by qPCR, immunoblots, and enzyme activity assays. We observed decreases in 3-NBA activation in the A549 NRF2 KO cell lines (53% reduction in A549 NRF2-Het cells and 82% reduction in A549 NRF2-KO cells) and 40-60% increases in 3-NBA bioactivation due to NRF2 activators in HBEC3-KT cells. Together, our data suggest that activation of the transcription factor NRF2 exacerbates carcinogen metabolism following exposure to diesel exhaust which may lead to an increase in 3-NBA-derived DNA adducts.

Exposure to polycyclic aromatic hydrocarbons derived from vehicle exhaust gas induces premature senescence in mouse lung fibroblast cells.

Long­term exposure to vehicle exhaust gas may lead to various age­associated disorders, including cardiovascular disease and cancer. Polycyclic aromatic hydrocarbons (PAHs) belong to an important class of carcinogens, which are released into the environment by vehicles and are detectable at high levels in Chinese urban areas. However, whether vehicle exhaust gas (EG), and in particular the PAHs derived from EG, are able to induce cell senescence remains unclear. In the present study, vehicle EG and pure PAHs were used as pollution sources to investigate the effects of long­term exposure to PAH on the cellular processes occurring in mouse lung fibroblast cells (mLFCs). Using cell proliferation and apoptosis assays, it was demonstrated that benzopyrene (BaP) suppressed the proliferation of mLFCs, and benzanthracene (BaA) and BaP induced cell apoptosis. Molecular analysis suggested that long­term exposure to BaA and BaP was able to increase the protein expression levels of p53, p21 and the apoptotic factors involved in the caspase cascade, including caspase­3 and ­9. Notably, the present study suggested that PAH exposure was able to promote cell senescence in mLFCs by activating the ATM serine/threonine kinase/H2A histone family member X pathway. The present study may provide novel insights into the underlying mechanism of vehicle EG and PAHs in promoting the development of age­associated diseases.

The processes associated with lipid peroxidation in human embryonic lung fibroblasts, treated with polycyclic aromatic hydrocarbons and organic extract from particulate matter.

Polycyclic aromatic hydrocarbons (PAHs) may cause lipid peroxidation via reactive oxygen species generation. 15-F2t-isoprostane (IsoP), an oxidative stress marker, is formed from arachidonic acid (AA) by a free-radical induced oxidation. AA may also be converted to prostaglandins (PG) by prostaglandin-endoperoxide synthase (PTGS) induced by NF-κB. We treated human embryonic lung fibroblasts (HEL12469) with benzo[a]pyrene (B[a]P), 3-nitrobenzanthrone (3-NBA) and extractable organic matter (EOM) from ambient air particulate matter <2.5 µm for 4 and 24 h. B[a]P and 3-NBA induced expression of PAH metabolising, but not antioxidant enzymes. The concentrations of IsoP decreased, whereas the levels of AA tended to increase. Although the activity of NF-κB was not detected, the tested compounds affected the expression of prostaglandin-endoperoxide synthase 2 (PTGS2). The levels of prostaglandin E2 (PGE2) decreased following exposure to B[a]P, whereas 3-NBA exposure tended to increase PGE2 concentration. A distinct response was observed after EOM exposure: expression of PAH-metabolising enzymes was induced, IsoP levels increased after 24-h treatment but AA concentration was not affected. The activity of NF-κB increased after both exposure periods, and a significant induction of PTGS2 expression was found following 4-h treatment. Similarly to PAHs, the EOM exposure was associated with a decrease of PGE2 levels. In summary, exposure to PAHs with low pro-oxidant potential results in a decrease of IsoP levels implying 'antioxidant' properties. For such compounds, IsoP may not be a suitable marker of lipid peroxidation.

Formation Mechanism of Benzo(a)pyrene: One of the Most Carcinogenic Polycyclic Aromatic Hydrocarbons (PAH).

The formation of polycyclic aromatic hydrocarbons (PAHs) is a strong global concern due to their harmful effects. To help the reduction of their emissions, a crucial understanding of their formation and a deep exploration of their growth mechanism is required. In the present work, the formation of benzo(a)pyrene was investigated computationally employing chrysene and benz(a)anthracene as starting materials. It was assumed a type of methyl addition/cyclization (MAC) was the valid growth mechanism in this case. Consequently, the reactions implied addition reactions, ring closures, hydrogen abstractions and intramolecular hydrogen shifts. These steps of the mechanism were computed to explore benzo(a)pyene formation. The corresponding energies of the chemical species were determined via hybrid density funcional theory (DFT), B3LYP/6-31+G(d,p) and M06-2X/6-311++G(d,p). Results showed that the two reaction routes had very similar trends energetically, the difference between the energy levels of the corresponding molecules was just 6.13 kJ/mol on average. The most stable structure was obtained in the benzo(a)anthracene pathway.

Functional impairment triggered by altertoxin II (ATXII) in intestinal cells in vitro: cross-talk between cytotoxicity and mechanotransduction.

Intestinal cells are able to continuously integrate response to multiple stimuli/stressors; these include the concomitant activation of "chemically driven" pathways, of paramount importance in the response to toxicants, as well as physical stimulation derived from motility. Altertoxin II (ATXII, 0.1, 1 and 10 µM), a mycotoxin produced by the food contaminant fungus Alternaria alternata was studied in HT-29 intestinal adenocarcinoma cells and in non-transformed intestinal epithelial cells, HCEC. One-hour incubation with ATXII was sufficient to trigger irreversible cytotoxicity in both cell types, as well as to modify cellular responses to concomitant pro-oxidant challenge (H2O2, 100-500 µM, DCF-DA assay) suggesting that even relatively short-time exposure of the intestinal cells could be sufficient to alter their functionality. Combination of ATXII (1 µM) with physical stimulation typical of the intestinal compartment (shear stress) revealed differential response of tumor-derived epithelial cells HT-29 in comparison to HCEC, in particular in the localization of the transcription factor Nrf2 (NF-E2-related factor 2). Moreover, ATXII reduced the migratory potential of HCEC as well as their membrane fluidity, but had no respective impact on HT-29 cells. Taken together, ATXII appeared to alter predominantly membrane functionality in HCEC thus hampering crucial functions for cellular motility/turnover, as well as barrier function of healthy intestinal cells and had very limited activity on the tumor counterparts.

7,12-Dimethylbenz[α]anthracene increases cell proliferation and invasion through induction of Wnt/ß-catenin signaling and EMT process.

7,12-Dimethylbenz[α]anthracene (DMBA) is a hazardous component present in polluted environments. DMBA has been used as an experimental tool for in vivo tumor formation owing to its carcinogenic effects, but the detailed molecular mechanism of DMBA has not been fully established. To comprehend the carcinogenic mechanism of DMBA, we explored its effects in the breast cancer cell lines, MCF-7 and MDA-MB-231, and the cervical cancer cell line, HeLa. Cell viability assay and measurement of a proliferation marker showed that DMBA markedly increased cancer cell proliferation. Furthermore, morphological observations and wound healing assays in nontumorigenic MCF-10A cells and trans-well invasion assays in cancer cells following DMBA treatment revealed that DMBA induced cell migration and invasion. To reveal the molecular mechanism of DMBA, we investigated the effects of DMBA on the epithelial-mesenchymal transition (EMT) process and Wnt/ß-catenin signaling, a critical pathway for cell proliferation that was reported to correlate with the EMT process, by using quantitative RT-PCR (qPCR), western blot analysis, and confocal microscopy. Consequently, we found that DMBA increased cancer cell proliferation and invasion through the promotion of EMT-inducing factors and ß-catenin. Especially, it was revealed in promoter activity assay using mutated luciferase vectors on transcription factor-binding sites that TWIST1 is promoted by DMBA through induction of STAT3-mediated promoter activation. To further elucidate the detailed mechanism of DMBA, we aimed to identify the key regulator of its carcinogenic action. DMBA was shown to significantly upregulate the expression of specificity protein 1 (Sp1), a transcription factor, and the carcinogenic effects of DMBA were blocked via the suppression or interruption of Sp1 activity. In conclusion, our data suggested that DMBA induced carcinogenic effects through activation of Wnt/ß-catenin signaling and the EMT process by upregulating Sp1 activity.

Delphinidin protects colon carcinoma cells against the genotoxic effects of the mycotoxin altertoxin II.

Alternaria spp. are ubiquitous molds that are able to produce toxic secondary metabolites which may contaminate food globally. One of those is the mycotoxin altertoxin II (ATX-II), a genotoxic and mutagenic compound. In recent years, different flavonoids that may co-occur with mycotoxins in food were demonstrated to temper toxic effects of molds, mostly through their anti-oxidant properties. Thus, in this study, we assessed the influence of the berry anthocyanidin delphinidin on the toxicity of ATX-II in HT-29 colon carcinoma cells. We performed coupled SRB/WST-1 cytotoxicity assays which revealed only weak antagonistic interactions, and single-cell gel electrophoresis ("comet") assays, where we observed a potent protective effect of delphinidin on the DNA-damaging properties of ATX-II. Furthermore, we investigated the mechanism for this interaction. In the DCF assay delphinidin was found to reduce intracellular oxidative stress levels, which might contribute partly to the latter protection. However, LC-MS experiments showed that co-incubation of the mycotoxin with either delphinidin or its potential degradation product phloroglucinol aldehyde significantly decreased ATX-II concentrations in aqueous solutions, indicating that a direct chemical reaction of ATX-II with these components is likely responsible for the observed loss of toxicity. Our results indicate that delphinidin - and possibly other anthocyanins as well - might play a role in the protection of the gut from Alternaria-induced genotoxicity.

Parthenolide attenuates 7,12-dimethylbenz[a]anthracene induced hamster buccal pouch carcinogenesis.

Over the decades, the survival rates for oral cancer have not improved despite development in novel diagnostic and therapeutic strategies. Therefore, the present study is aimed at investigating the chemopreventive potential of parthenolide in DMBA-induced hamster buccal pouch carcinogenesis. The hamsters were divided into 4 groups (n = 6/group). Group I was treated as control. Groups II and III were painted with a solution of 0.5% DMBA three times per week for 14 weeks on the left buccal pouches. In addition, group III were orally administrated with parthenolide 2 mg/kg b.w on days alternate to the DMBA application. Group IV received only parthenolide. At the end of 14th week all hamsters were sacrificed. Buccal tissues from all hamsters were evaluated for histopathology. Biochemical studies were carried out using plasma, liver, and buccal mucosa of control and experimental hamsters. Gene and protein expression studies of apoptotic markers p53, Bcl-2, and Bax were performed. The results showed 100% tumor formation and marked alterations in histopathology, status of detoxification enzymes, lipid peroxidation, and antioxidant profile in group II hamsters. Oral administration of parthenolide completely prevented tumor formation and significantly reduced the severity of histopathological changes in group III hamsters. The status of detoxification enzymes, lipid peroxidation, and antioxidants were significantly restored in parthenolide treated group compared to group II hamsters. The apoptotic gene p53 and antiapoptotic gene Bcl-2 were significantly down regulated; whereas, pro-apoptotic gene Bax was up regulated in group III hamsters compared to group II. The results of the present study suggest that parthenolide have potent chemopreventive, antioxidant, and apoptotic effect in DMBA-induced oral carcinogenesis.

p53 induction and cell viability modulation by genotoxic individual chemicals and mixtures.

The binding of the p53 tumor suppression protein to DNA response elements after genotoxic stress can be quantified by cell-based reporter gene assays as a DNA damage endpoint. Currently, bioassay evaluation of environmental samples requires further knowledge on p53 induction by chemical mixtures and on cytotoxicity interference with p53 induction analysis for proper interpretation of results. We investigated the effects of genotoxic pharmaceuticals (actinomycin D, cyclophosphamide) and nitroaromatic compounds (4-nitroquinoline 1-oxide, 3-nitrobenzanthrone) on p53 induction and cell viability using a reporter gene and a colorimetric assay, respectively. Individual exposures were conducted in the absence or presence of metabolic activation system, while binary and tertiary mixtures were tested in its absence only. Cell viability reduction tended to present direct correlation with p53 induction, and induction peaks occurred mainly at chemical concentrations causing cell viability below 80%. Mixtures presented in general good agreement between predicted and measured p53 induction factors at lower concentrations, while higher chemical concentrations gave lower values than expected. Cytotoxicity evaluation supported the selection of concentration ranges for the p53 assay and the interpretation of its results. The often used 80% viability threshold as a basis to select the maximum test concentration for cell-based assays was not adequate for p53 induction assessment. Instead, concentrations causing up to 50% cell viability reduction should be evaluated in order to identify the lowest observed effect concentration and peak values following meaningful p53 induction.

Dose-Dependent Response to 3-Nitrobenzanthrone Exposure in Human Urothelial Cancer Cells.

A product of incomplete combustion of diesel fuel, 3-nitrobenzanthrone (3-NBA), has been classified as a cancer-causing substance. It first gained attention as a potential urinary bladder carcinogen due to the presence of its metabolite in urine and formation of DNA adducts. The aim of the present study was to characterize the dose-response relationship of 3-NBA in human urothelial cancer cell line (RT4) exposed to concentrations ranging from 0.0003 µM (environmentally relevant) to 80 µM by utilizing toxicological and metabolomic approaches. We observed that the RT4 cells were capable of bioactivation of 3-NBA within 30 min of exposure. Activity measurements of various enzymes involved in the conversion of 3-NBA in RT4 cells demonstrated NAD(P)H:quinone oxidoreductase (NQO1) as the main contributor for its bioactivation. Moreover, cytotoxicity assessment exhibited an initiation of adaptive mechanisms at low dosages, which diminished at higher doses, indicating that the capacity of these mechanisms no longer suffices, resulting in increased levels of intracellular reactive oxygen species, reduced proliferation, and hyperpolarisation of the mitochondrial membrane. To characterize the underlying mechanisms of this cellular response, the metabolism of 3-NBA and metabolomic changes in the cells were analyzed. The metabolomic analysis of the cells (0.0003, 0.01, 0.08, 10, and 80 µM 3-NBA) showed elevated levels of various antioxidants at low concentrations of 3-NBA. However, at higher exposure concentrations, it appeared that the cells reprogrammed their metabolism to maintain the cell homeostasis via activation of pentose phosphate pathway (PPP).

Integration of cellular and molecular endpoints to assess the toxicity of polycyclic aromatic hydrocarbons in HepG2 cell line.

Polycyclic aromatic hydrocarbons (PAHs) are persistent pollutants present in the environment with known mutagenic and carcinogenic properties. In the present study the effects of exposure to single or multiple doses of benzo[a]anthracene (BaA), pyrene (Pyr), and 3 halogenated derivatives of these compounds (1-chloropyrene, 1-bromopyrene [1-BrPyr], and 7-chlorobenzo[a]anthracene [7-ClBaA]) were evaluated in a liver-derived human cell line (HepG2). Cytotoxicity as assessed by the classic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and neutral red assays showed a mild toxic effect in response to single or multiple dose exposure for up to 72 h, except for multiple dose exposure to BaA and 7-ClBaA (1 µM/d for 4 d) and single exposure to 10 µM BaA. Furthermore, selective mitochondrial and lysosomal toxicity was observed for Pyr and BaA series, respectively. To understand the underlying molecular mechanisms responsible for this effect, reactive oxygen species production, mitochondrial membrane depolarization, lysosomal pH, DNA fragmentation, and early and late apoptosis mediators were evaluated after exposure to single doses of the compounds. All compounds were able to trigger oxidative stress after 24 h as measured by catalase activity, and a good correlation was found between mitochondrial membrane depolarization, lysosomal pH increase, and MTT and neutral red assays. Evaluation of cell death mediators showed that caspase-3/7, but not annexin-V, pathways were involved in toxicity triggered by the studied compounds. The integration of all results showed that 1-BrPyr and BaA have a higher toxicity potential. Environ Toxicol Chem 2017;36:3404-3414. © 2017 SETAC.