Lance M. Hallberg

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.

Endogenous osteopontin promotes ozone-induced neutrophil recruitment to the lungs and airway hyperresponsiveness to methacholine

Inhalation of ozone (O₃), a common environmental pollutant, causes pulmonary injury, pulmonary inflammation, and airway hyperresponsiveness (AHR) in healthy individuals and exacerbates many of these same sequelae in individuals with preexisting lung disease. However, the mechanisms underlying these phenomena are poorly understood. Consequently, we sought to determine the contribution of osteopontin (OPN), a hormone and a pleiotropic cytokine, to the development of O₃-induced pulmonary injury, pulmonary inflammation, and AHR. To that end, we examined indices of these aforementioned sequelae in mice genetically deficient in OPN and in wild-type, C57BL/6 mice 24 h following the cessation of an acute (3 h) exposure to filtered room air (air) or O₃ (2 parts/million). In wild-type mice, O₃ exposure increased bronchoalveolar lavage fluid (BALF) OPN, whereas immunohistochemical analysis demonstrated that there were no differences in the number of OPN-positive alveolar macrophages between air- and O₃-exposed wild-type mice. O₃ exposure also increased BALF epithelial cells, protein, and neutrophils in wild-type and OPN-deficient mice compared with genotype-matched, air-exposed controls. However, following O₃ exposure, BALF neutrophils were significantly reduced in OPN-deficient compared with wild-type mice. When airway responsiveness to inhaled acetyl-β-methylcholine chloride (methacholine) was assessed using the forced oscillation technique, O₃ exposure caused hyperresponsiveness to methacholine in the airways and lung parenchyma of wild-type mice, but not OPN-deficient mice. These results demonstrate that OPN is increased in the air spaces following acute exposure to O₃ and functionally contributes to the development of O₃-induced pulmonary inflammation and airway and lung parenchymal hyperresponsiveness to methacholine.

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</=0.05), while colonic IGF-I mRNA levels were unchanged in aged AL rats. In aged CR rats, stomach IGFBP-3 mRNA levels decreased. Stomach and colonic IGF-IR mRNA levels declined with aging in AL and CR rats (P</=0.05). Colonic IGFBP-3 mRNA levels decreased significantly with aging in AL rats. There were no changes in colonic IGFBP-4 mRNA levels in aged AL or CR rats. Increased expression of stomach IGF-I, IGFBP-3 and IGFBP-4 in aged AL rats suggests that the stomach attempts to preserve IGF activity by increasing local expression of IGF-I and IGFBPs. Because the aging colon has a propensity to develop cancer, it may adapt to increased colonic IGF-I expression by reducing IGF-IR and IGFBP-3 expression. Additionally, CR lowers colonic IGF-I expression in aged rats (24 months) which may also be a protective adaptive mechanism.

DIFFERENTIAL INHIBITION OF MITOCHONDRIAL RESPIRATORY COMPLEXES BY INHALATION OF COMBUSTION SMOKE AND CARBON MONOXIDE, IN VIVO, IN THE RAT BRAIN

Combustion smoke contains gases and particulates, which act via hypoxia and cytotoxicity producing mechanisms to injure cells and tissues. While carbon monoxide (CO) is the major toxicant in smoke, its toxicity is exacerbated in the presence of other compounds. Here, we examined modulations of mitochondrial and cytosolic energy metabolism by inhalation of combustion smoke versus CO, in vivo, in the rat brain. Measurements revealed reduced activities of respiratory chain (RC) complexes, with greater inhibition by smoke than equivalent CO in ambient air. In the case of RC complex IV, inhibition by CO and smoke was similar—suggesting that complex IV inhibition is primarily by the action of CO. In contrast, inhibition of complexes I and III was greater by smoke. Increases in cytosolic lactate dehydrogenase and pyruvate kinase activities accompanied inhibition of RC complexes, likely reflecting compensatory increases in cytosolic energy production. Together, the data provide new insights into the mechanisms of smoke inhalation-induced perturbations of brain energetics, which impact neuronal function and contribute to the development of neuropathologies in survivors of exposures to CO and combustion smoke.

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.

Induction of deletion mutations by methoxyacetaldehyde in Chinese hamster ovary (CHO)-AS52 cells

We have reported previously that methoxyacetaldehyde (MALD), a metabolite of 2-methoxyethanol, induces gpt gene mutations in Chinese hamster ovary (CHO)-AS52 cells but not hprt gene mutations in the standard CHO-K1-BH4 cells. In addition, MALD induces chromosome aberrations in both CHO cell lines. The data presented suggest that MALD induces deletion-type mutations. In this study, we analyzed MALD-induced CHO-AS52 mutants for deletion-type mutations using the nested-polymerase chain reaction (nested-PCR) assay. Spontaneous CHO-AS52 mutants are used as untreated control. Ethylnitrosourea (ENU)-induced CHO-AS52 mutants are used as negative control for multilocus deletions since ENU is a potent inducer of point mutations. The results show that the frequency of MALD-induced mutants containing total deletion of the gpt gene is 42.4% which is 2.3-fold higher than that from spontaneous mutants (18.6%). The frequency of ENU-induced deletion mutation is 3%. The data substantiate our hypothesis that MALD induces major deletion mutations.

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.

1,3-Butadiene-induced mitochondrial dysfunction is correlated with mitochondrial CYP2E1 activity in Collaborative Cross mice

Cytochrome P450 2E1 (CYP2E1) metabolizes low molecular weight hydrophobic compounds, including 1,3-butadiene, which is converted by CYP2E1 to electrophilic epoxide metabolites that covalently modify cellular proteins and DNA. Previous CYP2E1 studies have mainly focused on the enzyme localized in the endoplasmic reticulum (erCYP2E1); however, active CYP2E1 has also been found in mitochondria (mtCYP2E1) and the distribution of CYP2E1 between organelles can influence an individuals response to exposure. Relatively few studies have focused on the contribution of mtCYP2E1 to activation of chemical toxicants. We hypothesized that CYP2E1 bioactivation of 1,3-butadiene within mitochondria adversely affects mitochondrial respiratory complexes I-IV. A population of Collaborative Cross mice was exposed to air (control) or 200ppm 1,3-butadiene. Subcellular fractions (mitochondria, DNA, and microsomes) were collected from frozen livers and CYP2E1 activity was measured in microsomes and mitochondria. Individual activities of mitochondrial respiratory complexes I-IV were measured using in vitro assays and purified mitochondrial fractions. In air- and 1,3-butadiene-exposed mouse samples, mtDNA copy numbers were assessed by RT-PCR, and mtDNA integrity was assessed through a PCR-based assay. No significant changes in mtDNA copy number or integrity were observed; however, there was a decrease in overall activity of mitochondrial respiratory complexes I, II, and IV after 1,3-butadiene exposure. Additionally, higher mtCYP2E1 (but not erCYP2E1) activity was correlated with decreased mitochondrial respiratory complex activity (in complexes I-IV) in the 1,3-butadiene-exposed (not control) animals. Together, these results represent the first in vivo link between mitochondrial CYP2E1 activity and mitochondrial toxicity.

Bis-butanediol-mercapturic acid (bis-BDMA) as a urinary biomarker of metabolic activation of butadiene to its ultimate carcinogenic species

Human carcinogen 1,3-butadiene (BD) undergoes metabolic activation to 3,4-epoxy-1-butene (EB), hydroxymethylvinyl ketone (HMVK), 3,4-epoxy-1,2-butanediol (EBD) and 1,2,3,4-diepoxybutane (DEB). Among these, DEB is by far the most genotoxic metabolite and is considered the ultimate carcinogenic species of BD. We have shown previously that BD-exposed laboratory mice form 8- to 10-fold more DEB-DNA adducts than rats exposed at the same conditions, which may be responsible for the enhanced sensitivity of mice to BD-mediated cancer. In the present study, we have identified 1,4-bis-(N-acetyl-L-cystein-S-yl)butane-2,3-diol (bis-BDMA) as a novel DEB-specific urinary biomarker. Isotope dilution high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry was employed to quantify bis-BDMA and three other BD-mercapturic acids, 2-(N-acetyl-L-cystein-S-yl)-1-hydroxybut-3-ene/1-(N-acetyl-L-cystein-S-yl)-2-hydroxy-but-3-ene (MHBMA, from EB), 4-(N-acetyl-L-cystein-S-yl)-1,2-dihydroxybutane (DHBMA, from HMVK) and 4-(N-acetyl-L-cystein-S-yl)-1,2,3-trihydroxybutane (THBMA, from EBD), in urine of confirmed smokers, occupationally exposed workers and BD-exposed laboratory rats. Bis-BDMA was formed in a dose-dependent manner in urine of rats exposed to 0-200 p.p.m. BD by inhalation, although it was a minor metabolite (1%) as compared with DHBMA (47%) and THBMA (37%). In humans, DHBMA was the most abundant BD-mercapturic acid excreted (93%), followed by THBMA (5%) and MHBMA (2%), whereas no bis-BDMA was detected. These results reveal significant differences in metabolism of BD between rats and humans.