Zemin Wang

Oxidative stress and oxidative damage in chemical carcinogenesis

Reactive oxygen species (ROS) are induced through a variety of endogenous and exogenous sources. Overwhelming of antioxidant and DNA repair mechanisms in the cell by ROS may result in oxidative stress and oxidative damage to the cell. This resulting oxidative stress can damage critical cellular macromolecules and/or modulate gene expression pathways. Cancer induction by chemical and physical agents involves a multi-step process. This process includes multiple molecular and cellular events to transform a normal cell to a malignant neoplastic cell. Oxidative damage resulting from ROS generation can participate in all stages of the cancer process. An association of ROS generation and human cancer induction has been shown. It appears that oxidative stress may both cause as well as modify the cancer process. Recently association between polymorphisms in oxidative DNA repair genes and antioxidant genes (single nucleotide polymorphisms) and human cancer susceptibility has been shown.

Evaluation of the Chronic Toxicity and Carcinogenicity of Perfluorohexanoic Acid (PFHxA) in Sprague-Dawley Rats

Perfluorohexanoic acid (PFHxA), a 6-carbon perfluoroalkyl (C6; CAS # 307-24-4), has been proposed as a replacement for the commonly used 8-carbon perfluoroalkyls: perfluorooctanoic acid and perfluorooctane sulfonate. PFHxA is not currently a commercial product but rather the ultimate degradation product of C6 fluorotelomer used to make C6 fluorotelomer acrylate polymers. It can be expected that, to a greater or lesser extent, the environmental loading of PFHxA will increase, as C6 fluorotelomer acrylate treatments are used and waste is generated. This article reports on a chronic study (duration 104 weeks) that was performed to evaluate the possible toxicologic and carcinogenic effects of PFHxA in gavage (daily gavage, 7 days per week) treated male and female Sprague-Dawley (SD) rats. In the current study, dosage levels of 0, 2.5, 15, and 100 mg/kg/day of PFHxA (males) and 5, 30, and 200 mg/kg/day of PFHxA (females) were selected based on a previous subchronic investigation. No effects on body weights, food consumption, a functional observational battery, or motor activity were observed after exposure to PFHxA. While no difference in survival rates in males was seen, a dose-dependent decrease in survival in PFHxA-treated female rats was observed. Hematology and serum chemistry were unaffected by PFHxA. PFHxA-related histologic changes were noted in the kidneys of the 200-mg/kg/day group females. Finally, there was no evidence that PFHxA was tumorigenic in male or female SD rats at any of the dosage levels examined.

Mechanistic Investigation of Toxaphene Induced Mouse Liver Tumors

Chronic exposure to toxaphene resulted in an increase in liver tumors in B6C3F1 mice. This study was performed to investigate the mode of action of toxaphene induced mouse liver tumors. Following an initial 14 day dietary dose range-finding study in male mice, a mechanistic study (0, 3, 32, and 320 ppm toxaphene in diet for 7, 14, and 28 days of treatment) was performed to examine the potential mechanisms of toxaphene induced mouse liver tumors. Toxaphene induced a significant increase in expression of constitutive androstane receptor (CAR) target genes (Cyp2b10, Cyp3a11) at 32 and 320 ppm toxaphene. aryl hydrocarbon receptor (AhR) target genes (Cyp1a1 and Cyp1a2) were slightly increased in expression at the highest toxaphene dose (320 ppm). No increase in peroxisome proliferator-activated receptor alpha activity or related genes was seen following toxaphene treatment. Lipid peroxidation was seen following treatment with 320 ppm toxaphene. These changes correlated with increases in hepatic DNA synthesis. To confirm the role of CAR in this mode of action, CAR knockout mice (CAR(-/-)) treated with toxaphene confirmed that the induction of CAR responsive genes seen in wild-type mice was abolished following treatment with toxaphene for 14 days. These findings, taken together with previously reported studies, support the mode of action of toxaphene induced mouse liver tumors is through a nongenotoxic mechanism involving primarily a CAR-mediated processes that results in an increase in cell proliferation in the liver, promotes the clonal expansion of preneoplastic lesions leading to adenoma formation.

Alkaline Comet Assay for Assessing DNA Damage in Individual Cells

Single‐cell gel electrophoresis, commonly called a comet assay, is a simple and sensitive method for assessing DNA damage at the single‐cell level. It is an important technique in genetic toxicological studies. The comet assay performed under alkaline conditions (pH >13) is considered the optimal version for identifying agents with genotoxic activity. The alkaline comet assay is capable of detecting DNA double‐strand breaks, single‐strand breaks, alkali‐labile sites, DNA‐DNA/DNA‐protein cross‐linking, and incomplete excision repair sites. The inclusion of digestion of lesion‐specific DNA repair enzymes in the procedure allows the detection of various DNA base alterations, such as oxidative base damage. This unit describes alkaline comet assay procedures for assessing DNA strand breaks and oxidative base alterations. These methods can be applied in a variety of cells from in vitro and in vivo experiments, as well as human studies.

Protective effects of antioxidants on acrylonitrile‐induced oxidative stress in female F344 rats

The induction of oxidative stress and damage appears to be involved in acrylonitrile induction of brain astrocytomas in rat. The present study examined the effects of dietary antioxidant supplementation on acrylonitrile‐induced oxidative stress and oxidative damage in rats in vivo. To assess the effects of antioxidants on biomarkers of acrylonitrile‐induced oxidative stress, female F344 rats were provided with diets containing vitamin E (0.05%), green tea polyphenols (GTP, 0.4%), N‐acetyl cysteine (NAC, 0.3%), sodium selenite (0.1mg/kg), and taurine (10g/kg) for 7 days, and then co‐administered with 0 and 100 ppm acrylonitrile in drinking water for 28 days. Significant increase in oxidative DNA damage in brain, evidenced by elevated 8OHdG levels, was seen in acrylonitrile‐exposed rats. Supplementation with vitamin E, GTP, and NAC reduced acrylonitrile‐induced oxidative DNA damage in brain while no protective effects were seen with the selenium or taurine supplementation. Acrylonitrile increased oxidative DNA damage, measured by the fpg‐modified alkaline Comet assay in rat WBCs, which was reduced by supplementation of Vitamin E, GTP, NAC, selenium, and taurine. In addition to stimulation of oxidative DNA damage, acrylonitrile triggered induction of pro‐inflammatory cytokines Tnfα, Il‐1β, and Ccl2, and the growth stimulatory cyclin D1 and cyclin D2 genes, which were effectively down‐regulated with antioxidant treatment. Antioxidant treatment also was able to stimulate the pro‐apoptotic genes Bad, Bax, and FasL and DNA repair genes Xrcc6 and Gadd45α. The results of this study support the involvement of oxidative stress in the development of acrylonitrile‐induced astrocytomas and suggest that antioxidants block acrylonitrile‐mediated damage through mechanisms that may involve in the suppression of inflammatory responses, inhibition of cell proliferation and stimulation of apoptosis.

Investigation of the mechanism of triclosan induced mouse liver tumors

&NA; Chronic dietary exposure to Triclosan (TCS) produced increased incidence of liver tumors in mice. The mechanism for liver tumor induction has been attributed to activation of either peroxisome proliferator activated receptor &agr; (PPAR&agr;) or constitutive androstane receptor (CAR). To further define the mechanism of TCS induced liver tumors, male CD‐1 and C57BL/6 mice were treated with TCS at 0, 10, 100 and 200 mg/kg diet/day for 14 or 28 days. In addition, a recovery group and positive control groups for CAR or PPAR&agr; activation with either phenobarbital or diethylhexyl‐phthalate were included in the 14‐day study. TCS induced a dose‐dependent increase in relative liver weight and centrilobular hypertrophy in both strains of mice. Hepatocyte DNA synthesis (BrdU labeling) was also increased in a dose‐related pattern. In comparison with previous studies, TCS induced a significant increase in CAR/PXR (Cyp2b10, Cyp3a11) and PPAR&agr; (Cyp4a10) responsive genes in both CD‐1 and C57BL/6 mice. The corresponding enzyme activity for CAR (7‐pentoxyresorufin‐O‐dealkylase) and PPAR&agr; (peroxisomal Acyl‐CoA oxidase) were also significantly increased in a similar fashion. Oxidative stress related genes Gpx1 and Aox1 were increased in the C57BL/6 but not in CD‐1 mice. The increases in gene expression and enzyme activities returned to control levels after 14‐day recovery. The present results demonstrate that both CAR and PPAR&agr; activation are involved in the TCS induced mouse liver tumor. HighlightsTriclosan produced a dose‐dependent increase in hepatocyte DNA synthesis in both CD‐1 and C57BL/6 mice.Triclosan activated both CAR and PPAR&agr; nuclear receptors in a dose‐responsive manner in CD‐1 and C57BL/6 mice.The triclosan mode of action of mouse liver tumors appears to be through both CAR and PPAR&agr; receptor mediated processes.

Cryopreservation of human blood for alkaline and Fpg-modified comet assay

Abstract The Comet assay is a reproducible and sensitive assay for the detection of DNA damage in eukaryotic cells and tissues. Incorporation of lesion specific, oxidative DNA damage repair enzymes (for example, Fpg, OGG1 and EndoIII) in the standard alkaline Comet assay procedure allows for the detection and measurement of oxidative DNA damage. The Comet assay using white blood cells (WBC) has proven useful in monitoring DNA damage from environmental agents in humans. However, it is often impractical to performance Comet assay immediately after blood sampling. Thus, storage of blood sample is required. In this study, we developed and tested a simple storage method for very small amount of whole blood for standard and Fpg-modified modified Comet assay. Whole blood was stored in RPMI 1640 media containing 10% FBS, 10% DMSO and 1 mM deferoxamine at a sample to media ratio of 1:50. Samples were stored at −20 °C and −80 °C for 1, 7, 14 and 28 days. Isolated lymphocytes from the same subjects were also stored under the same conditions for comparison. Direct DNA strand breakage and oxidative DNA damage in WBC and lymphocytes were analyzed using standard and Fpg-modified alkaline Comet assay and compared with freshly analyzed samples. No significant changes in either direct DNA strand breakage or oxidative DNA damage was seen in WBC and lymphocytes stored at −20 °C for 1 and 7 days compared to fresh samples. However, significant increases in both direct and oxidative DNA damage were seen in samples stored at −20 °C for 14 and 28 days. No changes in direct and oxidative DNA damage were observed in WBC and lymphocytes stored at −80 °C for up to 28 days. These results identified the proper storage conditions for storing whole blood or isolated lymphocytes to evaluate direct and oxidative DNA damage using standard and Fpg-modified alkaline Comet assay.

Mechanism of 1,3-Dichloropropene-Induced Rat Liver Carcinogenesis

1,3-Dichloropropene (1,3-D) is a soil fumigant used primarily for preplanting control of parasitic nematodes. In a previous chronic dietary exposure study, 1,3-D induced an increased incidence of hepatocellular adenomas in male rats at a dose of 25 mg/kg/day. Although the mechanism for tumor induction in the rat liver by 1,3-D has not been specifically elucidated, available data suggested that the observed liver tumorigenesis was through a nongenotoxic mode of action at the tumor promotion stage. Fischer 344 rats containing preneoplastic lesions were treated (via gavage) with 25 mg/kg/day 1,3-D or 80 mg/kg/day phenobarbital (PB) for 30 days and 60 days, or for 30 days followed by a 30-day recovery period (no compound exposure). Following treatment, placental form glutathione S-transferase (GSTP) positive and GSTP-negative liver focal lesions were quantitated as to size and number. 1,3-D treatment had no effect on GSTP-positive foci number or relative size but significantly increased the number, labeling index, and relative size of GSTP-negative focal lesions (identified by H and E staining) after 30 and 60 days of treatment. Following the 30-day recovery period, the number, labeling index, and relative size of the GSTP-negative lesions in 1,3-D-treated animals returned to control levels. As expected, PB treatment produced an increase in number and relative size of the GSTP-positive lesions. The results of this study are consistent with 1,3-D inducing liver carcinogenesis through a nongenotoxic mode of action by functioning as a tumor promoter specifically through induction of a non-GSTP staining focal hepatocyte population.

DNA damage in peripheral blood cells is associated with Alzheimer's disease (AD)-related plasma proteins in individuals at risk for progression to AD

correlations in their thicknesses. Here, we tested that hypothesis among patients with mild AD, patients with amnestic Mild Cognitive Impairment (aMCI), and age-matched healthy controls (HCs). Methods: We recruited 11 patients with AD, 12 with aMCI, and 18 HCs for clinical evaluation and MTL microstructural imaging: On an ultra-high field 7.0-Tesla MRI, we performed an oblique coronal T2-weighted sequence with in-plane resolution of 0.22 mm, permitting direct visual identification and measurement of MTL subfields CA1-SRLM, CA1 pyramidal layer (SP), dentate gyrus (DG)/CA3, and ERC using previously-established manual and semi-automated methods (Kerchner et al., 2012). Results: ERC and CA1-SRLM thicknesses correlated significantly in each of the three cohorts. Among HCs, this correlation was unique, and no other MTL structural covariance emerged. Only in the AD cohort, there was covariance between ERC and CA1-SP. DG/CA3 another synaptic target of ERC did not correlate with ERC in any cohort; only in the aMCI cohort, it correlated with both CA1-SRLM and CA1-SP. Absolute size differences were apparent in all MTL subfields between patients with AD and HCs, but not between patients with aMCI and HCs. As previously reported, delayed memory performance correlates with both ERC and CA1-SRLM thicknesses in mild AD (Kerchner et al., 2012), and we found the same phenomenon among our aMCI cohort but not HCs. Conclusions: ERC and CA1-SRLM two early targets of AD pathology demonstrate structural covariance among older adults regardless of cognitive status. In patients with AD, the cell body layer of CA1 joins in the correlation. A feature that distinguished aMCI and AD patients from HCs was a tight correlation between delayed memory performance and ERC and CA1-SRLM thicknesses, implicating these synaptically-connected, tau-vulnerable MTL subfields with a core symptom of the illness.

Effect of polyhexamethylene biguanide on rat liver.

Polyhexamethylene biguanide (PHMB), an amphiphilic polymeric biocide, increased liver tumor incidence in male and female rats at 1000 and 1500 mg/L in drinking water, but not at 500 mg/L in previous studies. In another study, PHMB administered in diet at 4000 mg/kg was negative for hepatocellular tumors. The present studies evaluated bioavailability and distribution of PHMB administered in drinking water and diet and possible modes of action (MOA). PHMB in drinking water was unpalatable during the first 3 days, resulting in markedly decreased food consumption and decreased body weight. Ki-67 labeling index was increased in hepatocytes and endothelial cells dose responsively with PHMB administered in drinking water but not diet. Vitamin E had no effect on this. There was no cytotoxicity by histopathology or serum enzymes, and no increase in cytokines TNFα, IL-1α or NF-κB. Focal iron deposition in sinusoidal lining cells was detected. Microarray analyses were non-contributory. No effect on CAR or PPARα activation was detected. 14C-PHMB administered at 500, 1000, or 1500 mg/L in the drinking water or 4000 mg/kg in the diet was nearly completely absorbed and excreted in urine, with some fecal excretion. The hypothesized MOA for liver tumors induced by PHMB in drinking water is: 1) severe dehydration and starvation because of unpalatability, followed by ingestion with rapid absorption and urinary excretion; 2) increased hepatocyte proliferation; and 3) induction of hepatocellular foci and tumors. The PHMB-induced rat hepatocellular tumors are unlikely to pose a human cancer risk. However, the actual MOA has not been determined.