Health effects of soy-biodiesel emissions: mutagenicity-emission factors.

CONTEXT: Soy biodiesel is the predominant biodiesel fuel used in the USA, but only a few, frequently conflicting studies have examined the potential health effects of its emissions. OBJECTIVE: We combusted petroleum diesel (B0) and fuels with increasing percentages of soy methyl esters (B20, B50 and B100) and determined the mutagenicity-emission factors expressed as revertants/megajoule of thermal energy consumed (rev/MJ(th)). MATERIALS AND METHODS: We combusted each fuel in replicate in a small (4.3-kW) diesel engine without emission controls at a constant load, extracted organics from the particles with dichloromethane, determined the percentage of extractable organic material (EOM), and evaluated these extracts for mutagenicity in 16 strains/S9 combinations of Salmonella. RESULTS: Mutagenic potencies of the EOM did not differ significantly between replicate experiments for B0 and B100 but did for B20 and B50. B0 had the highest rev/MJ(th), and those of B20 and B100 were 50% and ∼85% lower, respectively, in strains that detect mutagenicity due to polycyclic aromatic hydrocarbons (PAHs), nitroarenes, aromatic amines or oxidative mutagens. For all strains, the rev/MJ(th) decreased with increasing biodiesel in the fuel. The emission factor for the 16 EPA Priority PAHs correlated strongly (r(2 )= 0.69) with the mutagenicity-emission factor in strain TA100 + S9, which detects PAHs. CONCLUSIONS: Under a constant load, soy-biodiesel emissions were 50-85% less mutagenic than those of petroleum diesel. Without additional emission controls, petroleum and biodiesel fuels had mutagenicity-emission factors between those of large utility-scale combustors (e.g. natural gas, coal, or oil) and inefficient open-burning (e.g. residential wood fireplaces).

An evaluation of 25 selected ToxCast chemicals in medium-throughput assays to detect genotoxicity.

ToxCast is a multiyear effort to develop a cost-effective approach for the US EPA to prioritize chemicals for toxicity testing. Initial evaluation of more than 500 high-throughput (HT) microwell-based assays without metabolic activation showed that most lacked high specificity and sensitivity for detecting genotoxicants. Thus, EPA initiated a pilot project to investigate the use of standard genotoxicity endpoints using medium-throughput genotoxicity (MTG) assays in the context of a large testing program. Twenty-five chemicals were selected from the ToxCast program based in part on their known genotoxicity. The two MTG assays used were the Ames II(™) assay and 96-well In Vitro MicroFlow(®) Micronucleus (MN) assay. The Ames II assay showed a reasonable correlation with published Ames test data and industry submissions, though specificity was much better than sensitivity due to restraints on top concentrations as prescribed by ToxCast. Overall concordance was 73% both with and without metabolic activation. The flow MN assay had concordances of 71% and 58% with and without metabolic activation, respectively, when compared to published data and submissions. Importantly, a comparison of results without S9 from the MTG assays to an HT ToxCast p53 activation assay showed a fairly good degree of concordance (67%). The results reported here indicate that assays for genotoxicity endpoints can be conducted in a MT format and have the potential to add to the interpretation of results from large-scale testing programs such as EPA's ToxCast program. Inherent limitations such as the top concentrations used in large scale testing programs are discussed. Environ. Mol. Mutagen. 56:468-476, 2015. © 2014 Wiley Periodicals, Inc.

Cellular interactions and biological responses to titanium dioxide nanoparticles in HepG2 and BEAS-2B cells: role of cell culture media.

We showed previously that exposure of human lung cells (BEAS-2B) to TiO2 nanoparticles (nano-TiO2 ) produced micronuclei (MN) only when the final concentration of protein in the cell-culture medium was at least 1%. Nanoparticles localize in the liver; thus, we exposed human liver cells (HepG2) to nano-TiO2 and found the same requirement for MN induction. Nano-TiO2 also formed small agglomerates in medium containing as little as 1% protein and caused cellular interaction as measured by side scatter by flow cytometry and DNA damage (comet assay) in HepG2 cells. Nano-TiO2 also increased the activity of the inflammatory factor NFkB but not of AP1 in a reporter-gene HepG2 cell line. Suspension of nano-TiO2 in medium containing 0.1% protein was sufficient for induction of MN by the nanoparticles in either BEAS-2B or HepG2 cells as long the final concentration of protein in the cell-culture medium was at least 1%.

Catalase has a key role in protecting cells from the genotoxic effects of monomethylarsonous acid: a highly active metabolite of arsenic.

Although it is widely known that arsenic-contaminated drinking water causes many diseases, arsenic's exact mode of action (MOA) is not fully understood. Induction of oxidative stress has been proposed as an important key event in the toxic MOA of arsenic. The authors' studies are centered on identifying a reactive species involved in the genotoxicity of arsenic using a catalase (CAT) knockout mouse model that is impaired in its ability to breakdown hydrogen peroxide (H2 O2 ). The authors assessed the induction of DNA damage using the Comet assay following exposure of mouse Cat(+/) (+) and Cat(-) (/) (-) primary splenic lymphocytes to monomethylarsonous acid (MMA(III) ) to identify the potential role of H2 O2 in mediating cellular effects of this metalloid. The results showed that the Cat(-) (/) (-) lymphocytes are more susceptible to MMA(III) than the Cat(+/) (+) lymphocytes by a small (1.5-fold) but statistically significant difference. CAT activity assays demonstrated that liver tissue has approximately three times more CAT activity than lymphocytes. Therefore, Comet assays were performed on primary Cat(+/) (+) , Cat(+/) (-) , and Cat(-) (/) (-) hepatocytes to determine if the Cat(-) (/) (-) cells were more susceptible to MMA(III) than lymphocytes. The results showed that the Cat(-) (/) (-) hepatocytes exhibit higher levels of DNA strand breakage than the Cat(+/) (+) (approximately fivefold) and Cat(+/) (-) (approximately twofold) hepatocytes exposed to MMA(III) . Electron spin resonance using 5,5-dimethyl-1-pyrroline-N-oxide as the spin-trap agent detected the generation of ·OH via MMA(III) when H2 O2 was present. These experiments suggest that CAT is involved in protecting cells against the genotoxic effects of the ·OH generated by MMA(III) .

Effect of treatment media on the agglomeration of titanium dioxide nanoparticles: impact on genotoxicity, cellular interaction, and cell cycle.

The widespread use of titanium dioxide (TiO2) nanoparticles in consumer products increases the probability of exposure to humans and the environment. Although TiO2 nanoparticles have been shown to induce DNA damage (comet assay) and chromosome damage (micronucleus assay, MN) in vitro, no study has systematically assessed the influence of medium composition on the physicochemical characteristics and genotoxicity of TiO2 nanoparticles. We assessed TiO2 nanoparticle agglomeration, cellular interaction, induction of genotoxicity, and influence on cell cycle in human lung epithelial cells using three different nanoparticle-treatment media: keratinocyte growth medium (KGM) plus 0.1% bovine serum albumin (KB); a synthetic broncheoalveolar lavage fluid containing PBS, 0.6% bovine serum albumin and 0.001% surfactant (DM); or KGM with 10% fetal bovine serum (KF). The comet assay showed that TiO2 nanoparticles induced similar amounts of DNA damage in all three media, independent of the amount of agglomeration, cellular interaction, or cell-cycle changes measured by flow cytometry. In contrast, TiO2 nanoparticles induced MN only in KF, which is the medium that facilitated the lowest amount of agglomeration, the greatest amount of nanoparticle cellular interaction, and the highest population of cells accumulating in S phase. These results with TiO2 nanoparticles in KF demonstrate an association between medium composition, particle uptake, and nanoparticle interaction with cells, leading to chromosomal damage as measured by the MN assay.

Superoxide dismutase protects cells from DNA damage induced by trivalent methylated arsenicals.

Superoxide dismutase (SOD) catalyzes the conversion of superoxide to hydrogen peroxide. Heterozygous mice of strain B6;129S7-Sod1(tm1Leb)/J were obtained from Jackson Laboratories and bred to produce offspring that were heterozygous (+/Sod1(tm1Leb)), homozygous wild-type (+/+), and homozygous knockout (Sod1(tm1Leb) /Sod1(tm1Leb)) for the Cu/Zn superoxide dismutase (Sod1) gene. Splenocytes from these mice were exposed to several concentrations of either sodium arsenite (As3 [0-200 µM]), monomethylarsonous acid (MMA3 [0-10 µM]), or dimethylarsinous acid (DMA3 [0-10 µM]) for 2 hr. Cells were then examined for DNA damage using the alkaline single cell gel electrophoresis assay. Methyl methanesulfonate (MMS) was used as a positive control. Splenocytes from each of the three genotypes for Sod1 were equally sensitive to MMS and As3. However, at equimolar concentrations, DMA3 and MMA3 produced significantly more DNA damage in the homozygous knockout mouse splenocytes than in the splenocytes from the wild-type or heterozygous mice. These findings suggest that superoxide is involved either directly or indirectly in producing DNA damage in cells exposed to trivalent methylated arsenicals. These arsenicals may generate reactive oxygen species that damage DNA. This DNA damage may be a key factor in initiating cancer in vivo.

Organoarsenicals. Uptake, metabolism, and toxicity.

Arsenic is categorized by the WHO as the most significant environmental contaminant of drinking water due to the prevalence of geogenic contamination of groundwaters. Arsenic and the compounds which it forms are considered to be carcinogenic. The mechanism of toxicity and in particular of carcinogenicity of arsenic is still not well understood. The complexity originates from the fact that arsenic can form a rich variety of species, which show a wide variability in their toxicological behavior. The process of biomethylation was for many years regarded as a detoxification process; however, more recent research has indicated that the reverse is in fact the case. In this book chapter we give a summary of the current state of knowledge on the toxicities and toxicological mechanisms of organoarsenic species in order to evaluate the role and significance of these regarding their adverse effects on human health.

Cytogenetic insights into DNA damage and repair of lesions induced by a monomethylated trivalent arsenical.

Arsenic is a human carcinogen, and only recently animal models have been developed that are useful in investigating its carcinogenic mode of action (MOA). However, how arsenic induces cancer is still an open question. In a previous paper, we proposed a model detailing how arsenic might induce DNA lesions leading to cytogenetic damage [A.D. Kligerman, A.H. Tennant, Toxicol. Appl. Pharmacol. 222 (2007) 281-288]. In this model we hypothesized that arsenic does not induce chromosome damage via DNA adduction but induces short-lasting lesions from the action of reactive oxygen species (ROS). These lesions cause single-strand breaks (SSB) that induce chromosome breakage when treatment is in late G(1)- or S-phase. However, if treatment is confined to the G(0)- or early G(1)-phase of the cell cycle, it is predicted that little or no cytogenetic damage will result at the subsequent metaphase. Here, we describe the results from testing this model using monomethylarsonous acid (MMA(III)) and cytosine arabinoside (araC), a DNA chain terminator, to extend the time that DNA lesions remain open during repair to allow the lesions to reach S-phase or interact to form DNA exchanges that would lead to exchange aberrations at metaphase. The results of our study only partially confirmed our hypothesis. Instead, the results indicated that the lesions induced by MMA(III) are quickly repaired through base excision repair, that there is little chance for araC to extend the life of the lesions, and thus the DNA damage induced by arsenicals that leads to chromosome aberrations is very short lived.

Arsenate and dimethylarsinic acid in drinking water did not affect DNA damage repair in urinary bladder transitional cells or micronuclei in bone marrow.

Arsenic is a human skin, lung, and urinary bladder carcinogen, and may act as a cocarcinogen in the skin and urinary bladder. Possible modes of action of arsenic carcinogenesis/cocarcinogenesis include oxidative stress induction and inhibition of DNA damage repair. We investigated the effects of arsenic in drinking water on DNA damage repair in urinary bladder transitional cells and on micronucleus formation in bone marrow. F344 rats were given 100 ppm arsenate [As(V)] or dimethylarsinic acid [DMA(V)] in drinking water for 1 week. The in vivo repair of cyclophosphamide (CP)-induced DNA damage resulting from a single oral gavage of CP, and the in vitro repair of hydrogen peroxide (H(2)O(2))- or formaldehyde-induced DNA damage, resulting from adding H(2)O(2) or formaldehyde into cell medium, were measured by the Comet assay. DMA(V) effects were not observed on either CP-induced DNA damage induction or on DNA repair. Neither DMA(V) nor As(V) increased the H(2)O(2)- or formaldehyde-induced DNA damage, and neither inhibited the repair of H(2)O(2)-induced DNA damage. Neither DMA(V) nor As(V) increased the micronucleus frequency, nor did they elevate micronucleus frequency resulting from CP treatment above the level observed by the treatment with CP alone. These results suggest that arsenic carcinogenesis/cocarcinogenesis in the urinary bladder may not be via DNA damage repair inhibition. To our knowledge this is the first report of arsenic effects on DNA damage repair in the urinary bladder.

Measurement of DNA damage in rat urinary bladder transitional cells: improved selective harvest of transitional cells and detailed Comet assay protocols.

Urinary bladder transitional epithelium is the main site of bladder cancer, and the use of transitional cells to study carcinogenesis/genotoxicity is recommended over the use of whole bladders. Because the transitional epithelium is only a small fraction of the whole bladder, the alkaline single cell gel electrophoresis assay (Comet assay), which requires only a small number of cells per sample, is especially suitable for measuring DNA damage in transitional cells. However, existed procedures of cell collection did not yield transitional cells with a high purity, and pooling of samples was needed for Comet assay. The goal of this study was to develop an optimized protocol to evaluate DNA damage in the urinary bladder transitional epithelium. This was achieved by an enzymatic stripping method (trypsin-EDTA incubation plus gentle scraping) to selectively harvest transitional cells from rat bladders, and the use of the alkaline Comet assay to detect DNA strand breaks, alkaline labile sites, and DNA-protein crosslinks. Step by step procedures are reported here. Cells collected from a single rat bladder were sufficient for multiple Comet assays. With this new protocol, increases in DNA damage were detected in transitional cells after in vitro exposure to the positive control agents, hydrogen peroxide or formaldehyde. Repair of the induced DNA damage occurred within 4h. This indicated the capacity for DNA repair was maintained in the harvested cells. The new protocol provides a simple and inexpensive method to detect various types of DNA damage and to measure DNA damage repair in urinary bladder transitional cells.

Micronucleus studies in the peripheral blood and bone marrow of mice treated with jet fuels, JP-8 and Jet-A.

The potential adverse effects of dermal and inhalation exposure of jet fuels are important for health hazard evaluation in humans. The genotoxic potential of jet fuels, JP-8 and Jet-A, was investigated in an animal model. Mice were treated dermally with either a single or multiple applications of these jet fuels. Peripheral blood and bone marrow smears were prepared to examine the incidence of micronuclei (MN) in polychromatic erythrocytes (PCEs). In all experiments, using several different exposure regimens, no statistically significant increase in the incidence of MN was observed in the bone marrow and/or peripheral blood of mice treated with JP-8 or Jet-A when compared with those of untreated control animals. The data in mice treated with a single dose of JP-8 or Jet-A did not confirm the small but statistically significant increase in micronuclei reported in our previous study.

Induction of DNA-protein crosslinks by dichloromethane in a V79 cell line transfected with the murine glutathione-S-transferase theta 1 gene.

Dichloromethane (DCM) is considered a probable human carcinogen. Laboratory studies have shown an increased incidence of lung and liver cancer in mice but not in rats or hamsters. Despite the correlation between metabolism of DCM by the glutathione-S-transferase (GST) pathway and the occurrence of tumors in different species, the mechanism of tumor induction by DCM metabolites produced through the GST pathway remains unclear. In this study a V79 cell line stably transfected with the murine GST theta 1 gene (mGSTT1) was compared to the parent cell line (MZ) to determine how the construct affects DCM metabolism and the sensitivity of the cell line to DNA damage and cytotoxicity. V79 cells were treated with DCM (2.5-10mM) or formaldehyde (150-600muM) for 2h. Also, formaldehyde produced by V79 cytosol metabolism of DCM was measured spectrophotometrically. DNA damage and DNA-protein crosslinks were measured by the standard and proteinase K-modified alkaline single cell gel electrophoresis (SCG) assays. Cytotoxicity was assessed by trypan blue stain exclusion, the Live/Dead((R)) cell viability/cytotoxicity kit for animal cells, and the neutral red assay. After DCM treatment a significant concentration-dependent increase in tail moment in the V79 MZ cells was observed compared to a significant concentration-dependent decrease in tail moment in the V79 mGSTT1 cells. Post-incubation with proteinase K significantly increased DNA migrations in DCM-treated V79 mGSTT1 cells. DCM formed significantly higher levels of formaldehyde in the cytosol of the V79 mGSTT1 cells than in the cytosol of the V79 MZ cells. Results using the cytotoxicity assays were comparable using the trypan blue and Live/Dead((R)) assays, neither showing a difference in response between the two cell lines when exposed to either formaldehyde or DCM. These results indicate that V79 mGSTT1 can metabolize DCM to a genotoxic and cytotoxic metabolite, which is likely formaldehyde. This is the first time that the magnitude of the GSTT1 effect can be observed in mammalian cells without confounding caused by using cells with different genetic backgrounds.

Genotoxicity and metabolism of the source-water contaminant 1,1-dichloropropene: activation by GSTT1-1 and structure-activity considerations.

1,1-Dichloropropene (1,1-DCPe) is a contaminant of some source waters used to make drinking water. Because of this and the fact that no toxicological data were available for this compound, which is structurally similar to the rodent carcinogen 1,3-dichloropropene (1,3-DCPe), 1,1-DCPe was placed on the Contaminant Candidate List of the US Environmental Protection Agency. Consequently, we have performed a hazard characterization of 1,1-DCPe by evaluating its mutagenicity in the Salmonella assay and its DNA damaging (comet assay) and apoptotic (caspase assay) activities in human lymphoblastoid cells. In Salmonella, 1,1-DCPe was not mutagenic in strains TA98, TA100, TA1535, or TA104 +/-S9 mix. However, it was clearly mutagenic in strain RSJ100, which expresses the rat GSTT1-1 gene. 1,1-DCPe did not induce DNA damage in GSTT1-1-deficient human lymphoblastoid cells, and it induced apoptosis in these cells only at 5 mM. Consistent with its mutagenesis in RSJ100, 1,1-DCPe reacted with glutathione (GSH) in vitro, suggesting an addition-elimination mechanism to account for the detected GSH conjugate. 1,1-DCPe was approximately 5000 times more mutagenic than its ethene congener 1,1-dichloroethylene (1,1-DCE or vinylidene chloride). Neither 1,1-DCE nor 1,3-DCPe showed enhanced mutagenicity in strain RSJ100, indicating a lack of activation of these congeners by GSTT1-1. Thus, 1,1-DCPe is a base-substitution mutagen requiring activation by GSTT1-1, possibly involving the production of a reactive episulfonium ion. This bioactivation mechanism of 1,1-DCPe is different from that of its congeners 1,1-DCE and 1,3-DCPe. The presence of 1,1-DCPe in source waters could pose an ecological or human health risk. Occurrence data for 1,1-DCPe in finished drinking water are needed to estimate human exposure to, and possible health risks from, this mutagenic compound.

Methylated trivalent arsenicals as candidate ultimate genotoxic forms of arsenic: induction of chromosomal mutations but not gene mutations.

Arsenic is a prevalent human carcinogen whose mutagenicity has not been characterized fully. Exposure to either form of inorganic arsenic, As(III) or As(V), can result in the formation of at least four organic metabolites: monomethylarsonic acid, monomethylarsonous acid (MMA(III)), dimethylarsinic acid, and dimethylarsinous acid (DMA(III)). The methylated trivalent species, as well as some of the other species, have not been evaluated previously for the induction of chromosome aberrations, sister chromatid exchanges (SCE), or toxicity in cultured human peripheral blood lymphocytes; for mutagenicity in L5178Y/Tk(+/-) mouse lymphoma cells or in the Salmonella reversion assay; or for prophage-induction in Escherichia coli. Here we evaluated the arsenicals in these assays and found that MMA(III) and DMA(III) were the most potent clastogens of the six arsenicals in human lymphocytes and the most potent mutagens of the six arsenicals at the Tk(+/-) locus in mouse lymphoma cells. The dimethylated arsenicals were also spindle poisons, suggesting that they may be ultimate forms of arsenic that induce aneuploidy. Although the arsenicals were potent clastogens, none were potent SCE inducers, similar to clastogens that act via reactive oxygen species. None of the six arsenicals were gene mutagens in Salmonella TA98, TA100, or TA104; and neither MMA(III) nor DMA(III) induced prophage. Our results show that both methylated As(V) compounds were less cytotoxic and genotoxic than As(V), whereas both methylated As(III) compounds were more cytotoxic and genotoxic than As(III). Our data support the view that MMA(III) and DMA(III) are candidate ultimate genotoxic forms of arsenic and that they are clastogens and not gene mutagens. We suggest that the clastogenicity of the other arsenicals is due to their metabolism by cells to MMA(III) or DMA(III).

Comparison of the genotoxic activities of the K-region dihydrodiol of benzo[a]pyrene with benzo[a]pyrene in mammalian cells: morphological cell transformation; DNA damage; and stable covalent DNA adducts.

Benzo[a]pyrene (B[a]P) is the most thoroughly studied polycyclic aromatic hydrocarbon (PAH). Many mechanisms have been suggested to explain its carcinogenic activity, yet many questions still remain. K-region dihydrodiols of PAHs are metabolic intermediates depending on the specific cytochrome P450 and had been thought to be detoxification products. However, K-region dihydrodiols of several PAHs have recently been shown to morphologically transform mouse embryo C3H10T1/2CL8 cells (C3H10T1/2 cells). Because K-region dihydrodiols are not metabolically formed from PAHs by C3H10T1/2 cells, these cells provide a useful tool to independently study the mechanisms of action of PAHs and their K-region dihydrodiols. Here, we compare the morphological cell transforming, DNA damaging, and DNA adducting activities of the K-region dihydrodiol of B[a]P, trans-B[a]P-4,5-diol with B[a]P. Both trans-B[a]P-4,5-diol and B[a]P morphologically transformed C3H10T1/2 cells by producing both Types II and III transformed foci. The morphological cell transforming and cytotoxicity dose response curves for trans-B[a]P-4,5-diol and B[a]P were indistinguishable. Since morphological cell transformation is strongly associated with mutation and/or larger scale DNA damage in C3H10T1/2 cells, the identification of DNA damage induced in these cells by trans-B[a]P-4,5-diol was sought. Both trans-B[a]P-4,5-diol and B[a]P exhibited significant DNA damaging activity without significant concurrent cytotoxicity using the comet assay, but with different dose responses and comet tail distributions. DNA adduct patterns from C3H10T1/2 cells were examined after trans-B[a]P-4,5-diol or B[a]P treatment using 32P-postlabeling techniques and improved TLC elution systems designed to separate polar DNA adducts. While B[a]P treatment produced one major DNA adduct identified as anti-trans-B[a]P-7,8-diol-9,10-epoxide-deoxyguanosine, no stable covalent DNA adducts were detected in the DNA of trans-B[a]P-4,5-diol-treated cells. In summary, this study provides evidence for the DNA damaging and morphological cell transforming activities of the K-region dihydrodiol of B[a]P, in the absence of covalent stable DNA adducts. While trans-B[a]P-4,5-diol and B[a]P both induce morphological cell transformation, their activities as DNA damaging agents differ, both qualitatively and quantitatively. In concert with the morphological cell transformation activities of other K-region dihydrodiols of PAHs, these data suggest a new mechanism/pathway for the morphological cell transforming activities of B[a]P and its metabolites.