Jeffrey K. Wickliffe

Limitation of the MTT and XTT assays for measuring cell viability due to superoxide formation induced by nano-scale TiO2.

The reduction of the tetrazolium salts, MTT and XTT, is used to estimate cell viability and proliferation. However, superoxide can also reduce tetrazolium salts to produce the absorbant formazan end products. Evidence indicates that nano-TiO2 induces superoxide formation in different mammalian cells. Therefore, studies investigating the cytological effects of nano-TiO2 may encounter misleading results when using MTT/XTT to measure viability or proliferation. In this study, cell viabilities of Chinese hamster ovary cells were assayed using MTT, XTT and the trypan blue exclusion assay following exposure to nano-TiO2. In comparison to the trypan blue exclusion assay, the MTT and XTT assays inaccurately predicted cell toxicity or overestimated cell viability respectively. XTT, in particular, appears more sensitive to superoxide than MTT. The reduction rate of XTT is 1.5 times that of MTT and SOD inhibition of XTT is less effective than that of MTT, indicating that XTT is more reactive with O2- than MTT. Therefore, using XTT or MTT for measuring cell viability or proliferation may yield inaccurate results when conditions in cultured cell increase superoxide formation.

Nanoparticles: small and mighty

The properties of engineered nanomaterials and nanoparticles such as zinc oxide and titanium dioxide may differ substantially from naturally occurring materials and particles. Nanoparticles have unique physical properties making them ideal for use in various skin care products currently on the market. Nano‐preparations are currently under investigation as novel treatments of acne vulgaris, recurrent condyloma accuminata, atopic dermatitis, hyperpigmented skin lesions, and other non‐dermatologic diseases. Because of their increased surface area, nanoparticles have increased reactivity and a small size allowing for enhanced mobility through the human body and environment. As their use becomes more prevalent, nanoparticles are being scrutinized for their safety and long‐term effects. This review discusses the benefits of nanoparticles in dermatological therapies and skin care products as well as potential disadvantages and possible mechanisms of toxicity.

Single nucleotide polymorphisms of the DNA repair gene XPD/ERCC2 alter mRNA expression

Objectives Epidemiological studies documented associations between single nucleotide polymorphisms (SNPs) in the nucleotide excision repair gene XPD/ERCC2 and cancer risk. Little is known, however, about the underlying mechanisms for these associations. We explored a novel mechanism that could further explain the reported risk-modifying effect of these SNPs on disease susceptibility. Methods Using quantitative real-time polymerase chain reaction, we examined the relationship between three SNPs in the XPD gene (R156R in exon 6, D312N in exon 10 and K751Q in exon 23) and mRNA levels as a potential mechanism by which these SNPs could alter DNA repair capacity and affect disease risk. To further investigate the mechanism(s) by which these SNPs alter mRNA transcription levels, we performed a localized Mfold structure analysis on the mRNA sequence surrounding the studied SNPs. Results All three SNPs studied, alone and in combination, significantly decreased constitutive XPD mRNA levels (P<0.003) in lymphocytes of healthy subjects. The decrease in mRNA levels was significantly greater in smokers and was exacerbated by smoking duration and intensity. The decrease was more pronounced in older than in younger subjects. The R156R and the K751Q polymorphisms were predicted to alter mRNA secondary structure, indicating that these SNPs potentially affect local folding and mRNA stability. Conclusions Our results provide novel mechanistic explanations for epidemiological studies linking these SNPs to elevated cancer risk and emphasize the importance of comprehensively investigating the effect of both synonymous and nonsynonymous SNPs as risk modifiers by considering their potential effects on gene expression, protein translation and functions.

VARIATION OF MITOCHONDRIAL CONTROL REGION SEQUENCES OF STELLER SEA LIONS: THE THREE-STOCK HYPOTHESIS

Abstract Sequence variation in the mitochondrial DNA (mtDNA) control region was analyzed from 1,568 individuals representing nearly every rookery (n = 50) at which Steller sea lions (Eumetopias jubatus) are known to breed in significant numbers. Rookeries were grouped into regions and regions into stocks to examine structure at different spatial scales. Haplotype diversity (H = 0.9164 ± 0.0035) was high and nucleotide diversity (π = 0.00967 ± 0.00586) was moderate. No evidence was observed for significant genetic bottleneck effects. Previous studies of mtDNA recognized 2 stocks (eastern and western) and suggested the presence of 2 groups within the western stock. In this study, significant (P < 0.05) divergence of eastern stock (southeastern Alaska to California) animals from western stock animals was supported in analyses at all spatial scales. Likewise, rookeries and regions from Asia were found to be significantly different from all other western stock rookeries. This was most clearly demonstrated in regional comparisons. The Commander Islands rookery clearly associates with Alaskan western stock rookeries, not with the Asian rookeries. Within each of the 3 stocks there is significant isolation by distance among rookeries. This relationship does not hold for interstock comparisons, indicating that there are important barriers to gene flow among stocks. We recommend that the western stock be partitioned west of the Commander Islands, yielding a western stock that ranges from Prince William Sound west to the Commander Islands, and an Asian stock including rookeries from the Kamchatka Peninsula, Kuril Islands, and Sea of Okhotsk. The eastern stock remains unchanged and includes rookeries from southeastern Alaska through California.

Evaluation of Polycyclic Aromatic Hydrocarbons Using Analytical Methods, Toxicology, and Risk Assessment Research: Seafood Safety after a Petroleum Spill as an Example

Background: Polycyclic aromatic hydrocarbons (PAHs) are abundant and widespread environmental chemicals. They are produced naturally and through man-made processes, and they are common in organic media, including petroleum. Several PAHs are toxic, and a subset exhibit carcinogenic activity. PAHs represent a range of chemical structures based on two or more benzene rings and, depending on their source, can exhibit a variety of side modifications resulting from oxygenation, nitrogenation, and alkylation. Objectives: Here we discuss the increasing ability of contemporary analytical methods to distinguish not only different chemical structures among PAHs but also their concentrations in environmental media. Using seafood contamination following the Deepwater Horizon accident as an example, we identify issues that are emerging in the PAH risk assessment process because of increasing analytical sensitivity for individual PAHs, and we describe the paucity of toxicological literature for many of these compounds. Discussion: PAHs, including the large variety of chemically modified or substituted PAHs, are naturally occurring and may constitute health risks if human populations are exposed to hazardous levels. However, toxicity evaluations have not kept pace with modern analytic methods and their increased ability to detect substituted PAHs. Therefore, although it is possible to measure these compounds in seafood and other media, we do not have sufficient information on the potential toxicity of these compounds to incorporate them into human health risk assessments and characterizations. Conclusions: Future research efforts should strategically attempt to fill this toxicological knowledge gap so human health risk assessments of PAHs in environmental media or food can be better determined. This is especially important in the aftermath of petroleum spills. Citation: Wickliffe J, Overton E, Frickel S, Howard J, Wilson M, Simon B, Echsner S, Nguyen D, Gauthe D, Blake D, Miller C, Elferink C, Ansari S, Fernando H, Trapido E, Kane A. 2014. Evaluation of polycyclic aromatic hydrocarbons using analytical methods, toxicology, and risk assessment research: seafood safety after a petroleum spill as an example. Environ Health Perspect 122:6–9; http://dx.doi.org/10.1289/ehp.1306724

Chronic exposure to nanosized, anatase titanium dioxide is not cyto- or genotoxic to Chinese hamster ovary cells

Titanium dioxide nanoparticles (nano‐TiO2) are widely used in cosmetics, skin care products, paints, and water treatment processes. Disagreement remains regarding the safety of nano‐TiO2, and little epidemiological data is available to provide needed resolution. Most studies have examined effects using acute exposure experiments with relatively few studies using a chronic exposure design. We examined cyto‐ and genotoxicity in CHO‐K1 cells following 60 days of continuous exposure to defined levels of nano‐TiO2 (0, 10, 20, or 40 μg/ml). Oxidative stress increased in a concentration‐dependent manner in short‐ (2 days) and long‐term cultures, but long‐term cultures had lower levels of oxidative stress. The primary reactive oxygen species appeared to be superoxide, and ROS indicators were lowered with the addition of superoxide dismutase (SOD). No cyto‐ or genotoxic effects were apparent using the XTT, trypan‐blue exclusion, and colony‐forming assays for viability and the Comet and Hprt gene mutation assays for genotoxicity. Nano‐TiO2 increased the percentage of cells in the G2/M phase of the cell cycle, but this effect did not appear to influence cell viability or cell division. Cellular Ti content was dose‐dependent, but chronically exposed cells had lower amounts than acutely exposed cells. CHO cells appear to adapt to chronic exposure to nano‐TiO2 and detoxify excess ROS possibly through upregulation of SOD in addition to reducing particle uptake. Environ. Mol. Mutagen. 2011.

A targeted health risk assessment following the Deepwater Horizon oil spill: polycyclic aromatic hydrocarbon exposure in Vietnamese-American shrimp consumers.

Background: The Deepwater Horizon oil spill of 2010 prompted concern about health risks among seafood consumers exposed to polycyclic aromatic hydrocarbons (PAHs) via consumption of contaminated seafood. Objective: The objective of this study was to conduct population-specific probabilistic health risk assessments based on consumption of locally harvested white shrimp (Litopenaeus setiferus) among Vietnamese Americans in southeast Louisiana. Methods: We conducted a survey of Vietnamese Americans in southeast Louisiana to evaluate shrimp consumption, preparation methods, and body weight among shrimp consumers in the disaster-impacted region. We also collected and chemically analyzed locally harvested white shrimp for 81 individual PAHs. We combined the PAH levels (with accepted reference doses) found in the shrimp with the survey data to conduct Monte Carlo simulations for probabilistic noncancer health risk assessments. We also conducted probabilistic cancer risk assessments using relative potency factors (RPFs) to estimate cancer risks from the intake of PAHs from white shrimp. Results: Monte Carlo simulations were used to generate hazard quotient distributions for noncancer health risks, reported as mean ± SD, for naphthalene (1.8 × 10–4 ± 3.3 × 10–4), fluorene (2.4 × 10–5 ± 3.3 × 10–5), anthracene (3.9 × 10–6 ± 5.4 × 10–6), pyrene (3.2 × 10–5 ± 4.3 × 10–5), and fluoranthene (1.8 × 10–4 ± 3.3 × 10–4). A cancer risk distribution, based on RPF-adjusted PAH intake, was also generated (2.4 × 10–7 ± 3.9 × 10–7). Conclusions: The risk assessment results show no acute health risks or excess cancer risk associated with consumption of shrimp containing the levels of PAHs detected in our study, even among frequent shrimp consumers. Citation: Wilson MJ, Frickel S, Nguyen D, Bui T, Echsner S, Simon BR, Howard JL, Miller K, Wickliffe JK. 2015. A targeted health risk assessment following the Deepwater Horizon Oil Spill: polycyclic aromatic hydrocarbon exposure in Vietnamese-American shrimp consumers. Environ Health Perspect 123:152–159; http://dx.doi.org/10.1289/ehp.1408684

Persistence and Repair of Bifunctional DNA Adducts in Tissues of Laboratory Animals Exposed to 1,3-Butadiene by Inhalation

1,3-Butadiene (BD) is an important industrial and environmental chemical classified as a human carcinogen. The mechanism of BD-mediated cancer is of significant interest because of the widespread exposure of humans to BD from cigarette smoke and urban air. BD is metabolically activated to 1,2,3,4-diepoxybutane (DEB), which is a highly genotoxic and mutagenic bis-alkylating agent believed to be the ultimate carcinogenic species of BD. We have previously identified several types of DEB-specific DNA adducts, including bis-N7-guanine cross-links (bis-N7-BD), N(6)-adenine-N7-guanine cross-links (N(6)A-N7G-BD), and 1,N(6)-dA exocyclic adducts. These lesions were detected in tissues of laboratory rodents exposed to BD by inhalation ( Goggin et al. (2009) Cancer Res. 69 , 2479 -2486 ). In the present work, persistence and repair of bifunctional DEB-DNA adducts in tissues of mice and rats exposed to BD by inhalation were investigated. The half-lives of the most abundant cross-links, bis-N7G-BD, in mouse liver, kidney, and lungs were 2.3-2.4 days, 4.6-5.7 days, and 4.9 days, respectively. The in vitro half-lives of bis-N7G-BD were 3.5 days (S,S isomer) and 4.0 days (meso isomer) due to their spontaneous depurination. In contrast, tissue concentrations of the minor DEB adducts, N7G-N1A-BD and 1,N(6)-HMHP-dA, remained essentially unchanged during the course of the experiment, with an estimated t(1/2) of 36-42 days. No differences were observed between DEB-DNA adduct levels in BD-treated wild type mice and the corresponding animals deficient in methyl purine glycosylase or the Xpa gene. Our results indicate that DEB-induced N7G-N1A-BD and 1,N(6)-HMHP-dA adducts persist in vivo, potentially contributing to mutations and cancer observed as a result of BD exposure.

The L84F polymorphism in the O6-Methylguanine-DNA-Methyltransferase (MGMT) gene is associated with increased hypoxanthine phosphoribosyltransferase (HPRT) mutant frequency in lymphocytes of tobacco smokers.

Objectives O6-methylguanine-DNA-methyltransferase (MGMT) is a crucial DNA repair protein that removes DNA adducts formed by alkylating mutagens. Several coding single nucleotide polymorphisms (cSNPs) in the MGMT gene have been reported. Their biological significance, however, is not known. Methods We used a newly modified cloning HPRT mutant lymphocyte assay to test the hypothesis that inheritance of the L84F and I143V coding single nucleotide polymorphism in the MGMT gene is associated with increases in HPRT mutant frequency in lymphocytes of individuals exposed to alkylating agents. In addition, we expanded and sequenced 109 mutant clones to test the hypothesis that the mutation spectrum would shift to a larger percentage of base substitutions and G→A transition mutations in cells with L84F and I143 V coding single nucleotide polymorphisms. Results We observed no significant effect for the I143 V coding single nucleotide polymorphism on mutant frequency. In contrast, we observed a significant increase in mutant frequency (P<0.01) in lymphocytes from smokers with the 84F coding single nucleotide polymorphism compared with smokers homozygous for the referent L84 wild-type allele. A multiple regression analysis indicated that the mutant frequency increased significantly as a function of the 84F coding single nucleotide polymorphism and smoking, according to the model; mutant frequency (×10−5)=0.90+0.618 (84F polymorphism)+0.46 (smoking) with R2=0.22. Mutation spectra analysis revealed an apparent increase, which was short of statistical significance (P=0.08), in base substitutions in cells with the 84F polymorphism. Conclusions These new data suggest that the 84F coding single nucleotide polymorphism may alter the phenotype of the MGMT protein, resulting in suboptimal repair of O6-methylguanine lesions after exposure to alkylating agents.

Cadmium alters the formation of benzo[a]pyrene DNA adducts in the RPTEC/TERT1 human renal proximal tubule epithelial cell line.

Previously, we demonstrated the sensitivity of RPTEC/TERT1 cells, an immortalized human renal proximal tubule epithelial cell line, to two common environmental carcinogens, cadmium (Cd) and benzo[a]pyrene (B[a]P). Here, we measured BPDE-DNA adducts using a competitive ELISA method after cells were exposed to 0.01, 0.1, and 1 μM B[a]P to determine if these cells, which appear metabolically competent, produce BPDE metabolites that react with DNA. BPDE-DNA adducts were most significantly elevated at 1 μM B[a]P after 18 and 24 h with 36.34 ± 9.14 (n = 3) and 59.75 ± 17.03 (n = 3) adducts/108 nucleotides respectively. For mixture studies, cells were exposed to a non-cytotoxic concentration of Cd, 1 μM, for 24 h and subsequently exposed to concentrations of B[a]P for 24 h. Under these conditions, adducts detected at 1 μM B[a]P after 24 h were significantly reduced, 17.28 ± 1.30 (n = 3) adducts/108 nucleotides, in comparison to the same concentration at previous time points without Cd pre-treatment. We explored the NRF2 antioxidant pathway and total glutathione levels in cells as possible mechanisms reducing adduct formation under co-exposure. Results showed a significant increase in the expression of NRF2-responsive genes, GCLC, HMOX1, NQO1, after 1 μM Cd × 1 μM B[a]P co-exposure. Additionally, total glutathione levels were significantly increased in cells exposed to 1 μM Cd alone and 1 μM Cd × 1 μM B[a]P. Together, these results suggest that Cd may antagonize the formation of BPDE-DNA adducts in the RPTEC/TERT1 cell line under these conditions. We hypothesize that this occurs through priming of the antioxidant response pathway resulting in an increased capacity to detoxify BPDE prior to BPDE-DNA adduct formation.