Vernon E. Walker

Mitochondrial damage and DNA depletion in cord blood and umbilical cord from infants exposed in utero to Combivir.

Objective: Although most uninfected infants born to women infected with HIV-1 show no clinical evidence of mitochondrial compromise, mitochondrial dysfunction has been reported in children born to women receiving zidovudine and/or lamivudine during pregnancy. In this pilot study we examined mitochondrial integrity in HIV-1-uninfected infants born to HIV-1-infected women receiving Combivir during pregnancy. Design: Samples of umbilical cord and cord blood were obtained from HIV-1-uninfected infants born to either HIV-1-infected women receiving Combivir therapy during pregnancy (n = 10) or HIV-1-uninfected women (n = 9). Methods: Mitochondrial morphological integrity was examined in umbilical cords (n = 16) by electron microscopy and mtDNA quantity was determined in DNA from cord blood (n = 18) and umbilical cord (n = 18) by PCR-chemiluminescence immunoassay detection. Results: In umbilical cords from six of nine infants born to HIV-1-infected mothers taking Combivir moderate to severe mitochondrial morphological damage was observed (P = 0.011), while none of seven unexposed infants showed similar damage. Compared to unexposed infants, statistically significant mtDNA depletion was observed in umbilical cord (P = 0.006) and cord blood (P = 0.003) from drug-exposed infants. Conclusions: A cohort of HIV-1-uninfected Combivir-exposed infants with no clinical symptoms showed morphological and molecular evidence of mitochondrial damage.

A mouse model for the study of in vivo mutational spectra : sequence specificity of ethylene oxide at the hprt locus

We have developed an approach for determining mutational spectra in exon 3 of the hypoxanthine-guanine phosphoribosyl transferase (hprt) gene in splenic T-lymphocytes of B6C3F1 mice. Hprt- mutants from treated animals were isolated by culturing splenic T-cells in microtiter dishes containing medium supplemented with IL-2, concanavalin A, and 6-thioguanine. DNA was extracted from 6-thioguanine-resistant colonies and amplified by the polymerase chain reaction (PCR) using primers flanking the exon 3 region of hprt. Identification of samples containing mutant exon 3 sequences and purification of mutant DNA from contaminating wild-type hprt DNA was accomplished using denaturing gradient gel electrophoresis. Purified mutant sequences were then sequenced. This approach is being used to study the sequence specificity of ethylene oxide (ETO). 12-day-old mice were given single i.p. injections of 100 mg ETO/kg every other day or 30, 60, 90 or 120 mg ETO/kg daily for 5 days to achieve different cumulative doses of this compound. In mice exposed every other day, cumulative doses of 200, 600 and 900 mg ETO/kg produced average mutant frequencies of 15 +/- 12.8, 45 +/- 13.2, and 73 (70, 75) x 10(-6), respectively, 8 weeks after the first treatment. In mice exposed daily, cumulative doses of 150, 300, 450 and 600 mg ETO/kg produced average mutation frequencies of 4.2 +/- 10.4, 8.2 +/- 10.4, 11.1 +/- 1.0 and 15.5 +/- 10.7 x 10(-6), respectively, 20 weeks after the first treatment. The mutant fraction in control mice was less than 3 x 10(-6). 123 hprt- mutants from mice exposed to 600 or 900 mg ETO/kg were isolated and analyzed for mutations in exon 3. 18 were located in exon 3 (14.6%). DNA sequencing revealed that 11/18 mutations were base-pair substitutions at 8 different sites in exon 3. Four AT transversions, three AT transitions, two GC transversions, and two GC transitions were observed. Three of the substitutions (2 AT-->CG, 1 AT-->GC) occurred at one base (203) in a single animal. The remaining 7 mutations, isolated from 4 different animals, were the same +1 frameshift mutation in a run of 6 consecutive guanine bases (207-212) in exon 3. These results suggest the involvement of both modified guanine and adenine bases in ETO mutagenesis. The mouse T-cell cloning/sequencing assay for hprt described here represents a useful system for studying the molecular mechanism of chemically-induced mutation occurring in vivo at an endogenous gene.

1,3-Butadiene: Biomarkers and application to risk assessment

1,3-Butadiene (BD) is a known rodent and human carcinogen that is metabolized mainly by P450 2E1 to three epoxides, 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB) and 1,2-epoxy-3,4-butanediol (EB-diol). The individual epoxides vary up to 200-fold in their mutagenic potency, with DEB being the most mutagenic metabolite. It is important to understand the internal formation of the individual epoxides to assign the relative risk for each metabolite and to understand the molecular mechanisms responsible for major species differences in carcinogenicity. We have conducted extensive exposure-biomarker studies on mice, rats and humans. Using low exposures that range from current occupational levels to human exposures from tobacco smoke has provided evidence that mice are very different from humans, with mice forming ∼200 times more DEB than humans at exposures of 0.1-1.5ppm BD. While no gender differences have been noted in mice and rats for globin adducts or N-7 guanine adducts, female rats and mice had 2-3-fold higher Hprt mutations and DNA-DNA cross-links, suggesting a gender difference in DNA repair. Numerous molecular epidemiology studies have evaluated globin adducts and Hprt mutations, SCEs and chromosomal abnormalities. None of the blinded studies have shown evidence of human genotoxicity at current occupational exposures and studies of globin adducts have shown similar or lower formation of adducts in females than males. If one calculates the EB dose-equivalents for the three species, mice clearly differ from rats and humans, being ∼44 and 174 times greater than rats and humans, respectively. These data provide a scientific basis for improved risk assessment of BD.

Analysis of Diepoxide-Specific Cyclic N-Terminal Globin Adducts in Mice and Rats after Inhalation Exposure to 1,3-Butadiene

1,3-Butadiene is an important industrial chemical used in the production of synthetic rubber and is also found in gasoline and combustion products. It is a multispecies, multisite carcinogen in rodents, with mice being the most sensitive species. 1,3-Butadiene is metabolized to several epoxides that form DNA and protein adducts. Previous analysis of 1,2,3-trihydroxybutyl-valine globin adducts suggested that most adducts resulted from 3-butene-1,2-diol metabolism to 3,4-epoxy-1,2-butanediol, rather than from 1,2;3,4-diepoxybutane. To specifically examine metabolism of 1,3-butadiene to 1,2;3,4-diepoxybutane, the formation of the 1,2;3,4-diepoxybutane–specific adduct N,N-(2,3-dihydroxy-1,4-butadiyl)-valine was evaluated in mice treated with 3, 62.5, or 1250 ppm 1,3-butadiene for 10 days and rats exposed to 3 or 62.5 ppm 1,3-butadiene for 10 days, or to 1000 ppm 1,3-butadiene for 90 days, using a newly developed immunoaffinity liquid chromatography tandem mass spectrometry assay. In addition, 2-hydroxy-3-butenyl-valine and 1,2,3-trihydroxybutyl-valine adducts were determined. The analyses of several adducts derived from 1,3-butadiene metabolites provided new insight into species and exposure differences in 1,3-butadiene metabolism. Mice formed much higher amounts of N,N-(2,3-dihydroxy-1,4-butadiyl)–valine than rats. The formation of 2-hydroxy-3-butenyl-valine and N,N-(2,3-dihydroxy-1,4-butadiyl)–valine was similar in mice exposed to 3 or 62.5 ppm 1,3-butadiene, whereas 2-hydroxy-3-butenyl-valine was 3-fold higher at 1250 ppm. In both species, 1,2,3-trihydroxybutyl-valine adducts were much higher than 2-hydroxy-3-butenyl-valine and N,N-(2,3-dihydroxy-1,4-butadiyl)–valine. Together, these data show that 1,3-butadiene is primarily metabolized via the 3-butene-1,2-diol pathway, but that mice are much more efficient at forming 1,2;3,4-diepoxybutane than rats, particularly at low exposures. This assay should also be readily adaptable to molecular epidemiology studies on 1,3-butadiene-exposed workers

Using DNA and hemoglobin adducts to improve the risk assessment of butadiene.

The purpose of this paper is to review what we know about various biomarkers of butadiene in animal, human and in vitro studies, and to draw inferences from these data that impact on the accurate assessment of human risks for cancer. Studies comparing the DNA and hemoglobin adducts of butadiene with exposure, metabolism and genotoxicity have provided a great deal of insight that is applicable to biologically based risk assessment. First, the DNA and hemoglobin adduct data strongly support the conclusion that 3,4-epoxy-1,2-butanediol is the major electrophile available for binding to these macromolecules. Biomarker studies have also provided insight into the possibility of a sensitive population associated with the GSTT1 null genotype. While it is clear that lymphocytes from GSTT1 null individuals are more sensitive for the induction of sister chromatid exchanges (SCE) following in vitro exposure to 1,2,3,4-diepoxybutane, there was no such increase in SCE or other biomarkers of genotoxicity in workers exposed to 1-3 p.p.m. butadiene, regardless of GST genotype. The globin adduct data also demonstrate that there is roughly a tenfold range for interindividual differences in the metabolism of butadiene. This type of analysis represents an excellent means for providing scientific data for this critical determinant. Another useful application of hemoglobin adducts in risk assessment was demonstrated by regressing data for various endpoints for genotoxicity against that individuals biologically effective dose, thereby providing an independent mechanism for evaluation that excludes any possible confounding by inappropriate controls. Finally, biomarker studies have identified critical gaps in our knowledge that are needed for the accurate assessment of butadiene. Most notable of these is the lack of diepoxide-specific biomarkers in mice, rats and humans.

Molecular Dosimetry of 1,2,3,4-Diepoxybutane–Induced DNA-DNA Cross-Links in B6C3F1 Mice and F344 Rats Exposed to 1,3-Butadiene by Inhalation

1,3-Butadiene (BD) is an important industrial and environmental chemical classified as a human carcinogen based on epidemiologic studies in occupationally exposed workers and animal studies in laboratory rats and mice. BD is metabolically activated to three epoxides that can react with nucleophilic sites in biomolecules. Among these, 1,2,3,4-diepoxybutane (DEB) is considered the ultimate carcinogen due to its high genotoxicity and mutagenicity attributed to its ability to form DNA-DNA cross-links. Our laboratory has developed quantitative high-performance liquid chromatography-muESI(+)-tandem mass spectrometry methods for two DEB-specific DNA-DNA cross-links, 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD) and 1-(guan-7-yl)-4-(aden-1-yl)-2,3-butanediol (N7G-N1A-BD). This report describes molecular dosimetry analysis of these adducts in tissues of B6C3F1 mice and F344 rats exposed to a range of BD concentrations (0-625 ppm). Much higher (4- to 10-fold) levels of DEB-DNA cross-links were observed in mice compared with rats exposed to the same BD concentrations. In both species, bis-N7G-BD levels were 1.5- to 4-fold higher in the liver than in other tissues examined. Interestingly, tissues of female animals exposed to BD contained higher concentrations of bis-N7G-BD adducts than tissues of male animals, which is in accord with previously reported differences in tumor incidence. The molecular dosimetry data presented herein suggest that species and gender differences observed in BD-induced cancer are directly related to differences in the extent of BD metabolism to DEB. Furthermore, a rat model of sensitivity to BD may be more appropriate than a mouse model for assessing human risk associated with BD exposure, because rats and humans seem to be similar with respect to DEB formation.

Mechanistic Basis for Nonlinear Dose-Response Relationships for Low-Dose Radiation-Induced Stochastic Effects

The linear nonthreshold (LNT) model plays a central role in low-dose radiation risk assessment for humans. With the LNT model, any radiation exposure is assumed to increase ones risk of cancer. Based on the LNT model, others have predicted tens of thousands of deaths related to environmental exposure to radioactive material from nuclear accidents (e.g., Chernobyl) and fallout from nuclear weapons testing. Here, we introduce a mechanism-based model for low-dose, radiation-induced, stochastic effects (genomic instability, apoptosis, mutations, neoplastic transformation) that leads to a LNT relationship between the risk for neoplastic transformation and dose only in special cases. It is shown that nonlinear dose-response relationships for risk of stochastic effects (problematic nonlethal mutations, neoplastic transformation) should be expected based on known biological mechanisms. Further, for low-dose, low-dose rate, low-LET radiation, large thresholds may exist for cancer induction. We summarize previously published data demonstrating large thresholds for cancer induction. We also provide evidence for low-dose-radiation-induced, protection (assumed via apoptosis) from neoplastic transformation. We speculate based on work of others (Chung 2002) that such protection may also be induced to operate on existing cancer cells and may be amplified by apoptosis-inducing agents such as dietary isothiocyanates.

In vivo mutagenicity of ethylene oxide at the hprt locus in T-lymphocytes of B6C3F1 lacI transgenic mice following inhalation exposure

Ethylene oxide (EO) is a direct-acting alkylating agent with the potential to induce cytogenetic alterations, mutations, and cancer. In the present study, the in vivo mutagenicity of EO at the hypoxanthine guanine phosphoribosyltransferase (hprt) locus of T-lymphocytes was evaluated following inhalation exposure of male B6C3F1 lacI transgenic mice. For this purpose, groups of male Big Blue mice at 6-8 (n = 4/group) and 8-10 (n = 5/group) weeks of age were exposed to 0, 50, 100, or 200 ppm EO for 4 weeks (6 h/day, 5 days/week). At necropsy, T-cells were isolated from thymus and/or spleen and cultured in the presence of concanavalin A, IL-2, and 6-thioguanine [Skopek, T.R., V.E. Walker, J.E. Cochrane et al. (1992) Proc. Natl. Acad. Sci. USA, 89, 7866-7870]. The time course for expression of hprt-negative lymphocytes in thymus was determined in mice necropsied 2 h, 2 weeks, and 8 weeks after exposure to 200 ppm EO. The dose-response for hprt mutant T-cells in thymus and spleen was defined in mice necropsied 2 and 8 weeks post-exposure, respectively. The hprt mutant frequency (Mf) in thymus of exposed mice was increased 2 h after exposure and reached a maximum of 7.5 +/- 0.9 x 10(-6) (average Mf +/- SE) at 2 weeks post-exposure, compared with 2.3 +/- 0.8 x 10(-6) in thymus of control mice. Dose-related increases in hprt Mfs were found in thymus from mice exposed to 100 and 200 ppm EO. In addition, a nonlinear dose-dependent increase in hprt Mfs was observed in splenic T-cells, with greater mutagenic efficiency (mutations per unit dose) found at higher concentrations than at lower concentrations of EO. Average induced Mfs (i.e. induced Mf = treatment Mf - background Mf) in splenic T-cells were 1.6, 4.6, and 11.9 x 10(-6) following exposures to 50, 100, or 200 ppm EO, respectively, while the average control Mf value was 2.2 +/- 0.3 x 10(-6). In aliquots of lymphocytes (both B- and T-cells) isolated from spleen for analysis of lacI mutations in the same animals, only two of three EO-exposed mice at the 200 ppm exposure level demonstrated an elevated lacI Mf and these elevations were apparently due to the in vivo replication of preexisting mutants and not due to the induction of new mutations associated with EO exposure [Sisk, S., L.J. Pluta, K.G. Meyer and L. Recio (1996) Mutation Res., submitted]. These data demonstrate that repeated inhalation exposures to high concentrations of EO produce dose-related increases in mutations at the hprt locus of T-lymphocytes in male lacI transgenic mice of B6C3F1 origin.

Persistence of mitochondrial toxicity in hearts of female B6C3F1 mice exposed in utero to 3′-azido-3′-deoxythymidine

Cardiac toxicity has been associated with HIV infection and exposure to nucleoside reverse transcriptase inhibitors (NRTIs), but the role of the latter in the development of cardiac disease of HIV-infected patients is uncertain. To investigate the cardiotoxicity of transplacentally administered zidovudine (AZT) or AZT plus lamivudine (3TC) in the absence of HIV infection, we evaluated several biomarkers of cardiac mitochondrial structure and cardiac structure and function in a B6C3F1 mouse model. In utero exposure to AZT-3TC resulted in ultrastructural pathology, loss of mitochondria, and altered echocardiographic measurements in newborn mice. Cardiac pathology and dysfunction persisted into the adult life of female mice exposed in utero to AZT, as evidenced by significant dose-dependent heart enlargement, clusters of atypical mitochondria and myofibril alterations, significantly increased cytochrome c oxidase activity, and significantly higher numbers of mutations in mitochondrial tRNA genes compared with unexposed controls at 18 to 24 mo of age. These data led to the hypothesis that the long-term pathology of perinatal exposure to these NRTIs is related to persistent mitochondrial DNA mutations in cardiac tissue; that is, the primary damage during drug treatment is mutational (as opposed to affecting polymerase γ and/or other mitochondrial elements) and leads over time to delayed, progressive cardiotoxicity.

Detection of cyclophosphamide-induced mutations at the Hprt but not the lacI locus in splenic lymphocytes of exposed mice.

The relative sensitivities and specificities of the endogenous Hprt gene and the lacI transgene as mutational targets were evaluated in splenic lymphocytes from male standard B6C3F1 mice (only Hprt assayed) and from lacI transgenic B6C3F1 mice treated at 6–7 weeks‐ of‐age with the indirect‐acting agent, cyclophosphamide (CP). To define the effects of the time elapsed since CP treatment on Hprt mutant frequencies (Mfs), nontransgenic mice were given single i.p. injections of 25 mg CP/kg or vehicle (PBS) alone and then necropsied 2, 4, 6, 8, or 10 weeks after treatment. Peak Mfs were found at 6 weeks postexposure, with mean Mf values ranging from 2.27 to 3.27 × 10–5 using two different lots of CP in standard packaging (compared with mean control Mf values of 0.14 to 0.26 × 10–5 in various experiments). To determine the dose response for Hprt Mfs, nontransgenic mice were given single doses of 0, 12.5, 25, 50, or 100 mg CP/kg and necropsied 4 weeks postexposure. These treatments produced a supralinear dose response curve for CP‐induced Hprt Mfs. Based on these experiments, CP mutagenicities at Hprt and lacI were compared in transgenic mice treated with 0, 25, or 100 mg CP/kg (using another lot of CP in ISOPAC® bottles; Sigma) and necropsied 6 weeks later. There was a significant increase in Hprt Mfs in treated transgenic mice (100 mg CP/kg: 0.75 ± 0.09 × 10–5; 25 mg CP/kg: 0.39 ± 0.05 × 10–5) versus controls (0.10 ± 0.01 × 10–5); however, the Mfs in lacI of lymphocytes from the same CP‐treated animals were not significantly different from controls (100 mg CP/kg: 9.4 ± 1.1 × 10–5; 25 mg CP/kg: 6.7 ± 0.8 × 10–5; control: 7.7 ± 0.7 × 10–5). Hprt mutational spectra data in CP‐treated transgenic and nontransgenic mice were different from those of control mice, whereas the spectra of mutations in lacI of lymphocytes from Big Blue® transgenic mice were not significantly changed after CP treatment. These data indicate that, under these treatment conditions, CP‐induced mutations in splenic lymphocytes were detectable in the Hprt gene but not the lacI transgene of this nontarget tissue for CP‐induced cancer. Environ. Mol. Mutagen. 34:167–181, 1999