Part 3. Assessment of genotoxicity and oxidative stress after exposure to diesel exhaust from U.S. 2007-compliant diesel engines: report on 1- and 3-month exposures in the ACES bioassay.

Human health hazards due to diesel exhaust (DE*) exposure have been associated with both solvent and combustion components. In the past, diesel engine exhaust components have been linked to increased mutagenicity in cultures of Salmonella typhimurium and mammalian cells (Tokiwa and Ohnishi 1986). In addition, DE has been shown to increase both the incidence of tumors and the induction of 8-hydroxy-deoxyguanosine adducts (8-OHdG) in ICR mice (Ichinose et al. 1997). Furthermore, DE is composed of a complex mixture of polycyclic aromatic hydrocarbons (PAHs) and particulates. One such PAH, 3-nitrobenzanthrone (3-NBA), has been identified in DE and found in urban air. 3-NBA has been observed to induce micronucleus formation in DNA of human hepatoma cells (Lamy et al. 2004). The purpose of the current research, which is part of the Advanced Collaborative Emissions Study (ACES), a multidisciplinary program being carried out by the Health Effects Institute and the Coordinating Research Council, is to determine whether improvements in the engineering of heavy-duty diesel engines reduce the oxidative stress and genotoxic risk associated with exposure to DE components. To this end, the genotoxicity and oxidative stress of DE from an improved diesel engine was evaluated in bioassays of tissues from Wistar Han rats and C57BL/6 mice exposed to DE. Genotoxicity was measured as strand breaks using an alkaline-modified comet assay. To correlate possible DNA damage found by the comet assay, measurement of DNA-adduct formation was evaluated by a competitive enzyme-linked immunosorbent assay (ELISA) to determine the levels of free 8-OHdG found in the serum of the animals exposed to DE. 8-OHdG is a specific modified base indicating an oxidative type of DNA damage to DNA nucleotides. In addition, a thiobarbituric acid reactive substances (TBARS) assay was used to assess oxidative stress and damage in the form of lipid peroxidation in the hippocampus region of the brains of DE-exposed animals. Results from the comet assay showed no significant differences in rats between the control and exposed groups (P = 0.53, low exposure; P = 0.92, medium exposure; P = 0.77, high exposure) after 1 month of DE exposure. There were no differences between sexes in the responses of rats to these exposures. Likewise, there were no significant differences found after 3 months of exposure. Similarly, no significant differences were found between the mice exposed for 1 and 3 months to DE, nor were any differences found between sexes. Measurements of 8-OHdG in both mice and rats showed no significant difference among DE exposure groups (P = 0.46, mice; P = 0.86, rats). In mice, measured 8-OHdG was lower in the 3-month group than the 1-month group. In rats, the inverse was true. In mice, no significant differences in the levels of lipid peroxidation, as measured by TBARS, were found between the controls and DE exposure groups (P = 0.92), nor were there any differences between sexes. In rats, comparisons between the control and low-exposure groups approached significance, but no significant differences were found between the other DE exposure groups. Additionally, in rats, there were no significant differences between the 1- and 3-month DE exposure groups.

Thermal properties of ration components as affected by moisture content and water activity during freezing.

Beef roast with vegetables is an example of a meal, ready-to-eat (MRE) ration entrée. It is a mixture of meat, potato, mushroom, and carrot with a gravy sauce. The thermal properties of each component were characterized in terms of freezing point, latent heat, freezable and unfreezable water contents, and enthalpy during freezing using differential scanning calorimetry. Freezing and thawing curves and the effect of freezing and thawing cycles on thermal properties were also evaluated. The freezing points of beef, potato, mushroom, and sauce were all in the range of -5.1 to -5.6 degrees C, but moisture content, water activity, latent heat, freezable and unfreezable water contents, and enthalpy varied among these components. Freezing temperature greatly affected the unfrozen water fraction. The unfreezable water content (unfrozen water fraction at -50 degrees C) of ration components was in the range of 8.2% to 9.7%. The freezing and thawing curves of vegetables with sauce differed from those of beef but took similar time to freeze or thaw. Freezing and thawing cycles did not greatly affect the thermal properties of each component. Freezing point and latent heat were reduced by decreasing moisture content and water activity of each component. Water activity was proportionally linear to freezing point at a(w) > 0.88, and moisture content was proportionally linear to freezable water content in all ration components. Water was not available for freezing when moisture content was reduced to 28.8% or less. This study indicates that moisture content and water activity are critical factors affecting thermal behavior of ration components during freezing.

Benzo(a)pyrene activates L1Md retrotransposon and inhibits DNA repair in vascular smooth muscle cells.

Benzo(a)pyrene (BaP) modulates vascular smooth muscle cells (vSMCs) from a quiescent to proliferative phenotype, a shift associated with activation of L1Md retrotransposon [K.P. Lu, K.S. Ramos, Biochem. Biophys. Res. Commun. 253 (1998) 828-833]. The present studies were conducted to evaluate L1Md activation profiles in murine vSMCs treated with BaP or its oxidative metabolites, and to screen for possible insertional mutations into p53 and retinoblastoma (RB) genes. We also sought to examine the profile of DNA damage and repair in BaP-treated vSMCs. Northern analysis revealed that BaP (0. 03-3microM), and its major reactive 7,8-diol metabolite (0. 03-3microM), activate L1Md gene in a concentration-dependent manner. Two other metabolites, 3-OH BaP and 3,6-BaP quinone (0.03-3microM), as well as hydrogen peroxide (25-75microM) also activated L1Md. No insertional mutations into either p53 or RB genes were observed in vSMCs treated with BaP in vitro, although a slight elevation of p53 mRNA was observed as early as 4h after chemical challenge. Treatment of vSMCs with 3 or 30microM BaP for 4h increased unscheduled DNA synthesis (UDS) 1.4- and 2.5-fold, respectively. Challenge with 0. 3microM BaP for 24h inhibited DNA repair capacity in vSMCs for up to 48h. These results demonstrate that BaP and its oxidative metabolites activate L1Md retrotransposon in vSMCs, which coupled to DNA damage and inhibition of DNA repair are part of the atherogenic response elicited by BaP and related hydrocarbons.

Effects of aging and caloric restriction on IGF-I, IGF-I receptor, IGFBP-3 and IGFBP-4 gene expression in the rat stomach and colon.

The purpose of this study was to determine the effects of aging and caloric restriction (CR) on insulin-like growth factor-I (IGF-I), IGF-I receptor (IGF-IR), IGF-binding protein-3 (IGFBP-3) and IGFBP-4 expression in the stomach and colon of male Fischer 344 rats. Stomach and colonic RNA were prepared from ad libitum (AL) fed or long-term CR rats. Stomach IGF-I, IGFBP-3 and IGFBP-4 mRNA levels increased significantly (P

Rapid SINE-mediated detection of cisplatin:DNA adduct formation in vitro and in vivo in blood.

Cisplatin (cis-dichlorodiammine platinum II) is one of the most effective antitumor agents to date. Its usefulness is limited, however, by toxicity to healthy tissues, most notably, its nephrotoxicity. To maximize the chemotherapeutic potential of cisplatin and minimize its adverse effects, it is imperative to monitor formation of cisplatin:DNA adducts throughout treatment. We developed a novel, highly sensitive, SINE (Short Interspersed DNA Element)-mediated. PCR-based assay for detection of cisplatin adducts in vitro and in vivo, in DNA from mouse blood cells. The assay relies on the abundance, dispersion and conservation of SINEs in mammalian genomes. The B1 elements at a copy number of 50,000-80,000 are the most abundant SINEs in the mouse genome. Due to their strong sequence conservation, primers complementary to the B1 consensus sequence anneal to the majority of their targets in the genome and allow simultaneous amplification of long random segments of genomic DNA. Thus, in conjunction with the fact that cisplatin adducts block the progression of thermostable polymerase, B1 element-anchored PCR makes a sensitive tool for assessing the overall integrity of the transcribed regions in the mouse genome. The high sensitivity of the assay allows detection of DNA damage at the low cisplatin dosage of 1-8 mg/kg that is considered as sub-chemotherapeutic in experimental animal models. The sensitivity range therefore, makes this assay suitable for the development of predictive correlation for both, the efficacy of treatment as well as induction of nephrotoxicity.

Short interspersed DNA element-mediated detection of UVB-induced DNA damage and repair in the mouse genome, in vitro, and in vivo in skin.

We report a sensitive, SINE (Short Interspersed DNA Element)-mediated, PCR-based, DNA damage detection assay. Here, the SINE assay is used for detection of UVB-induced DNA damage and repair in cultured mouse cells and in vivo, in mouse skin. The unique feature of the SINE assay is its ability to support simultaneous amplification of multiple, random segments of genomic DNA. This can be accomplished due to the remarkable abundance, dispersion and conservation of SINEs in mammalian genomes. The most abundant SINEs in the mouse genome are the B1 elements, at a copy number of 50,000-80,000. Due to their strong sequence conservation, primers complementary to the B1 consensus sequence anneal to the majority of their targets in the genome. Consequently, long segments of genomic DNA located between pairs of B1 elements are efficiently amplified by PCR. Thus, in conjunction with the fact that many types of DNA adducts form blocks for thermostable polymerase, the B1 element anchored PCR makes a sensitive and versatile tool for assessing the overall integrity of the transcribed regions in mouse genome. We measured UVB-dose (0.1-3 kJ m-2) dependent formation of photoproducts in DNA from cultured cells, and after 20 h observed a substantial removal of damage at doses lower or equal to 0.6 kJ m-2. The sensitivity of detection of UVB-photoproducts formation and repair was compared to that of the conventional, single locus-targeting QPCR. Using the SINE assay we also have shown the distribution of UVB and UVC induced DNA adducts at a single nucleotide resolution within the B1 elements in mouse DNA. Lastly, we demonstrated that the sensitivity of the SINE assay is adequate for measurement of UVB-dose (1-6 kJ m-2) dependent formation and subsequent removal of photoproducts in vivo, in mouse skin.

Abnormal DNA repair activities in lymphocytes of workers exposed to 1,3-butadiene.

Exposure to high concentrations of butadiene has been shown to cause cancer among exposed workers. We have conducted a biomarker study to elucidate whether current butadiene exposure conditions are hazardous to workers. Twenty-four workers exposed consistently to butadiene were matched with 19 co-workers who had much less contact with butadiene and who served as our controls. In the standard cytogenetic assay, there was no difference in chromosome aberration frequencies between the exposed and control groups. In the challenge assay, the exposed group shows a consistent, but non-significant, increase in chromosome aberrations indicating some abnormality in DNA repair response. The observed dicentric frequency in the challenge assay (indicative of abnormal repair of damaged chromosomes) is significantly correlated with a butadiene metabolite, 1,2-dihydroxy-4-(N-acetylcysteinyl)butane, in urine (r = 0.52; p = 0.04). Furthermore, cigarette smokers had consistently abnormal repair response compared with non-smokers for both the control and exposed groups. A small subset of the studied workers were evaluated for toxicant-induced DNA repair deficiency using an independent cat-host cell reactivation (CAT-HCR) assay. When cigarette smokers and non-smokers were combined in our analysis, we observed that the exposed group (n = 9) had a significant reduction of DNA repair activities (p = 0.009) compared with the control group (n = 6). Cigarette smoking contributed significantly to the effect as exposed smokers (n = 4) had a significant reduction in DNA repair activities (p = 0.04) compared with exposed non-smokers. The results from the two independently conducted assays support each other and confirm the previously reported abnormal DNA repair response in another group of butadiene workers. In conclusion, our data indicates that exposure to environmental toxicants, such as butadiene, can cause DNA repair defects. Therefore, the current butadiene exposure conditions are still hazardous to workers. However, our data indicates that butadiene is not a potent genotoxic agent. Furthermore, the butadiene-induced effect is significantly enhanced by the cigarette smoking habit.

Measurement of DNA repair deficiency in workers exposed to benzene.

We hypothesize that chronic exposure to environmental toxicants can induce genetic damage causing DNA repair deficiencies and leading to the postulated mutator phenotype of carcinogenesis. To test our hypothesis, a host cell reactivation (HCR) assay was used in which pCMVcat plasmids were damaged with UV light (175, 350 J/m2 UV light), inactivating the chloramphenicol acetyltransferase reporter gene, and then transfected into lymphocytes. Transfected lymphocytes were therefore challenged to repair the damaged plasmids, reactivating the reporter gene. Xeroderma pigmentosum (XP) and Gaucher cell lines were used as positive and negative controls for the HCR assay. The Gaucher cell line repaired normally but XP cell lines demonstrated lower repair activity. Additionally, the repair activity of the XP heterozygous cell line showed intermediate repair compared to the homozygous XP and Gaucher cells. We used HCR to measure the effects of benzene exposure on 12 exposed and 8 nonexposed workers from a local benzene plant. Plasmids 175 J/m2 and 350 J/m2 were repaired with a mean frequency of 66% and 58%, respectively, in control workers compared to 71% and 62% in exposed workers. Conversely, more of the exposed workers were grouped into the reduced repair category than controls. These differences in repair capacity between exposed and control workers were, however, not statistically significant. The lack of significant differences between the exposed and control groups may be due to extremely low exposure to benzene (< 0.3 ppm), small population size, or a lack of benzene genotoxicity at these concentrations. These results are consistent with a parallel hprt gene mutation assay.