Miriam C. Poirier

Chemical Carcinogenesis: From Animal Models To Molecular Models In One Decade

During the last decade, progress in chemical carcinogenesis research has been substantial, and understanding the cellular changes and molecular causes of initiation, promotion, and malignant conversion appears to be within reach. Cancer begins as a carcinogen-induced genetic change in a single cell. The interaction of a particular carcinogen with specific genetic sites results, in part, from selectivity of metabolically activated carcinogens for particular nucleosides or gene sequences. In turn, modification of the molecular structure at specific genetic loci will have tissue-specific and species-specific consequences dependent on the expression of a particular gene, its sequence, and the function of the gene product in the target cell. It is likely that inactivation of regulatory regions, genomic rearrangements, and point mutations in coding sequences all can result in an altered cell phenotype. The rasH gene (and perhaps other members of the ras gene family) appears to be a common target for coding sequence mutations in the initiation of carcinogenesis in several organ sites and species by specific carcinogens. Whatever genetic mechanisms are involved, an initiated cell phenotype common to many epithelial cell types is observed. Initiated cells have an altered program of terminal differentiation, are resistant to cytotoxic substances or show altered requirements for specific growth factors or nutrients. These cells would have a selective growth advantage in cytostatic or cytotoxic situations or under conditions favoring terminal differentiation. Tumor promoters, some acting through specific cellular receptors, produce a tissue environment conductive to the selective clonal outgrowth of the initiated cell population resulting in a clinically evident premalignant lesion. The tissue specificity for most promoters depends on the ability of a particular agent to produce the selective conditions required for the initiated phenotype of that organ. At the molecular level, phorbol ester tumor promoters bind to and activate protein kinase C and transduce signals through this second-messenger pathway. Heterogeneity in the species of protein kinase C molecule expressed by normal and initiated epidermal cells could account for the differential response pattern observed in these cell types during skin tumor promotion. Malignant conversion of benign tumors requires further genetic changes in the tumor cell. Such changes could result from inherent instability in the genome of initiated cells, from spontaneous mutations more likely to occur in the expanding population of proliferating benign tumor cells, or by additional exposure to exogenous genotoxic agents.(ABSTRACT TRUNCATED AT 400 WORDS)

Methods of DNA adduct determination and their application to testing compounds for genotoxicity

At the International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC (March 25–26, 1999), a working group considered the uses of DNA adduct determination methods for testing compounds for genotoxicity. When a drug or chemical displays an unusual or inconsistent combination of positive and negative results in in vitro and in vivo genotoxicity assays and/or in carcinogenicity experiments, investigations into whether or not DNA adducts are formed may be helpful in assessing whether or not the test compound is a genotoxin. DNA adduct determinations can be carried out using radiolabeled compounds and measuring radioactive decay (scintillation counting) or isotope ratios (accelerator mass spectrometry) in the isolated DNA. With unlabeled compounds adducts may be measured by 32P‐postlabeling analysis of the DNA, or by physicochemical methods including mass spectrometry, fluorescence spectroscopy, or electrochemical detection, or by immunochemical methods. Each of these approaches has different strengths and limitations, influenced by sensitivity, cost, time, and interpretation of results. The design of DNA binding studies needs to be on a case‐by‐case basis, depending on the compounds profile of activity. DNA purity becomes increasingly important the more sensitive, and less chemically specific, the assay. While there may be adduct levels at which there is no observable biological effect, there are at present insufficient data on which to set a threshold level for biological significance. Environ. Mol. Mutagen. 35:222–233, 2000

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.

Mitochondrial Toxicity in Fetal Erythrocebus patas Monkeys Exposed Transplacentally to Zidovudine Plus Lamivudine

This study was designed to investigate fetal mitochondrial toxicity in Erythrocebus patas monkeys exposed in utero to zidovudine (AZT) and lamivudine (3TC), and taken at term. Pregnant patas monkeys were given a daily dose of 40 mg AZT (86% of the human daily dose, based on body weight), for the last 10 weeks (50%) of gestation, and a daily dose of 24 mg 3TC (84% of the human daily dose, based on body weight) for the last 4 weeks of gestation. At term, AZT was found to be incorporated into fetal mitochondrial DNA from skeletal muscle, liver, kidney, and placenta. By transmission electron microscopy (EM) drug-exposed fetal cardiac and skeletal muscle cells showed mitochondrial membrane compromise, mitochondrial proliferation, and damaged sarcomeres, while mitochondria in brain cerebrum and cerebellum were morphologically normal. Substantial depletion of oxidative phosphorylation (OXPHOS) Complex I specific activities was observed in heart (87% reduction in mean, p = 0.02) and skeletal muscle (98% reduction in mean, p = 0.002) from drug-exposed fetuses, compared to unexposed fetuses. In addition Complex IV activity was highly depleted (85% reduction in mean, p = 0.004) in skeletal muscle from the drug-exposed fetuses (p = 0.004). Brain cerebrum and cerebellum showed no statistically significant OXPHOS changes with drug exposure. Mitochondrial DNA quantity was substantially depleted (>50%) in heart, skeletal muscle, cerebellum, and cerebrum from drug-exposed fetuses compared to unexposed controls. Overall, the data indicate that significant mitochondrial damage was observed at birth in monkey fetuses exposed in utero to AZT plus 3TC in a human-equivalent dosing protocol.

Dose response effects of 2-acetylaminofluorene on DNA damage, cytotoxicity, cell proliferation and neoplastic conversion in rat liver

This study measured the effect of precise doses of 2-acetylaminofluorene (AAF) in inducing DNA damage, functional changes and neoplastic conversion in rat liver. Groups of male F344 rats at 9 weeks of age were exposed to cumulative doses of 0.5 or 2.0 mmol AAF per kg body weight given by gavage daily 5 days per week over an 8-week period and maintained with no further exposure for up to 8 weeks. Administration of AAF resulted in the formation of N-deoxyguanosin-(8-yl)-2-aminofluorene in liver DNA in relationship to dose. In centrilobular hepatocytes the zone of glutamine synthetase-expressing cells was reduced by exposure. By 8 weeks, but not at 4 weeks, the higher of the two doses of AAF provoked an increase in cell proliferation measured by immunohistochemical incorporation of bromodeoxyuridine. Altered hepatocellular foci expressing the placental form of glutathione transferase were induced by the high dose of AAF at 4 weeks, but not at the low dose. At 8 weeks the incidence of foci at the high dose was 79 times that induced by the low dose. These foci were highly proliferative. In animals exposed to AAF for 8 weeks and maintained for 4 weeks with no exposure, DNA adducts decreased by 80% and cell proliferation subsided by 80%, although the glutamine synthetase zone remained diminished. After discontinuation of AAF, the number of foci diminished by 50% and their proliferation subsided by 80% at 4 weeks, indicating a phenotypic reversion of many foci. With this protocol of administration of precise doses of AAF, we have established non-linearity of effects and a lack of correlation between DNA adduct formation and induction of cellular lesions. We suggest that doses in the range of those reported can be used to study the contribution of epigenetic and genotoxic effects in carcinogenesis and to study threshold events.

Transplacental genotoxicity of combined Antiretroviral nucleoside analogue therapy in Erythrocebus patas monkeys

Summary: Antiretroviral nucleoside analogue drugs are a major constituent of highly active antiretroviral therapy (HAART). the most advanced form of treatment for HIV‐1 infection. Currently, HAART combinations that include zidovudine (ZDV) and lamivudine (3TC) are highly effective in preventing HIV‐1 vertical transmission; most children are born with no evident adverse clinical effects. However, ZDV is a moderately strong transplacental carcinogen in mice, and potential long‐term consequences of fetal exposure to most HAART combinations remain unknown. To model human transplacental ZDV and 3TC exposures, experiments were performed in Erythrocebus patas monkeys given human‐equivalent drug exposure protocols. Pregnant monkeys were dosed with either no drug (n = 2), 40.0 mg ZDV/d (about 6 mg/kg body weight/d) for the last 50% (10 weeks) of gestation (n = 3), or with the same regimen of ZDV plus 24.0 mg 3TC/d (about 3.6 mg/kg body weight/d) for the last 20% (4 weeks) of gestation (n = 3). Multiple fetal organs were examined at term for DNA incorporation of ZDV and 3TC using two separate radioimmunoassays (RIAs). Values for ZDV‐DNA incorporation were similar in fetuses exposed to ZDV alone and those exposed to ZDV plus 3TC. Values for 3TC‐DNA in fetal organs were greater than or equal to values for ZDV‐DNA, indicating that the total DNA damage sustained by fetuses exposed to both drugs was at least double that observed in fetuses exposed to ZDV alone. Telomere shortening, determined by Southern blot with a telomeric probe, was observed in most organs of the three animals exposed in utero to ZDV plus 3TC. No telomere shortening was evident in the unexposed fetuses, and occasional telomere shortening was found in fetuses exposed to ZDV alone. Overall, these studies demonstrate that monkey fetuses exposed in utero to the combination ZDV plus 3TC sustain a higher level of drug‐DNA incorporation and show evidence of more telomere damage than monkey fetuses exposed to ZDV alone.

Polycyclic Aromatic Hydrocarbon (PAH) Exposure and DNA Adduct Semi-Quantitation in Archived Human Tissues

Polycyclic aromatic hydrocarbons (PAHs) are combustion products of organic materials, mixtures of which contain multiple known and probable human carcinogens. PAHs occur in indoor and outdoor air, as well as in char-broiled meats and fish. Human exposure to PAHs occurs by inhalation, ingestion and topical absorption, and subsequently formed metabolites are either rendered hydrophilic and excreted, or bioactivated and bound to cellular macromolecules. The formation of PAH-DNA adducts (DNA binding products), considered a necessary step in PAH-initiated carcinogenesis, has been widely studied in experimental models and has been documented in human tissues. This review describes immunohistochemistry (IHC) studies, which reveal localization of PAH-DNA adducts in human tissues, and semi-quantify PAH-DNA adduct levels using the Automated Cellular Imaging System (ACIS). These studies have shown that PAH-DNA adducts concentrate in: basal and supra-basal epithelium of the esophagus, cervix and vulva; glandular epithelium of the prostate; and cytotrophoblast cells and syncitiotrophoblast knots of the placenta. The IHC photomicrographs reveal the ubiquitous nature of PAH-DNA adduct formation in human tissues as well as PAH-DNA adduct accumulation in specific, vulnerable, cell types. This semi-quantative method for PAH-DNA adduct measurement could potentially see widespread use in molecular epidemiology studies.

Preferential binding of cisplatin to mitochondrial DNA of Chinese hamster ovary cells

Some chemical carcinogens localize preferentially in mitochondrial DNA (mtDNA) when compared with genomic DNA (gDNA). Here we compare the ability of cisplatin (cis-diamminedichloroplatinum[II]) to induce DNA adducts in both genomic and mtDNA of Chinese hamster ovary (CHO) cells in culture. Cytotoxicity was examined by cell survival 4, 8 and 24 h after exposure to 50 microM cisplatin. Cisplatin-DNA adducts were measured in DNA from nuclear and mitochondrial fractions by dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA), a sensitive competitive microtiter-based immunoassay utilizing antiserum elicited against cisplatin-modified DNA. An additional comparison of cisplatin-DNA binding in both compartments was performed by immunoelectron microscopy using the cisplatin-DNA antiserum and colloidal gold. DELFIA analysis of cisplatin-DNA adducts in gDNA and mtDNA showed a six-fold higher incorporation of drug into mtDNA as compared to gDNA. Morphometric studies of colloidal gold distribution in photomicrographs of CHO cells showed mtDNA to contain a four-fold higher concentration of cisplatin as compared to nuclear DNA. Therefore, both methods demonstrated a preferential binding of cisplatin to mtDNA versus gDNA.

Biomonitoring of human genotoxicity induced by complex occupational exposures

Sensitivity, specificity and correlations among several biomarkers for monitoring occupational exposure to complex mixtures of genotoxic agents were assessed in occupational environments in Hungarian study populations. The studies have been focused on DNA adduct formation, urinary metabolites, mutations and micronuclei induced by exposures to complex organic mixtures. In two Hungarian aluminium plants, increased DNA adduct and 1-hydroxypyrene (1-OH-PY) levels were observed in workers as compared to controls. However, no association between the biomarker levels was evident on an individual basis. In Hungarian garage mechanics, DNA adduct determinations did not show increased genotoxic exposure as compared to the controls. However, ambient air measurements, significantly enhanced 1-OH-PY levels, and slightly enhanced frequency of micronuclei indicated increased polycyclic aromatic hydrocarbon (PAH) exposure in the garages, as compared to the general environment. In a Hungarian vulcanizing plant, DNA adduct determinations and 1-OH-PY did not show significantly elevated exposure levels as compared to controls. The glycophorin A (GPA) somatic mutation assay was also negative for this occupational exposure. The results support previous observations of a lack of correlation between DNA adducts detectable by 32P-postlabelling and those measured by the PAH-DNA immunoassay in the same DNA sample. These studies also demonstrate a lack of close correlation between levels of DNA adducts and urinary 1-OH-PY in the same individual.

Fetal Patas Monkeys Sustain Mitochondrial Toxicity As a Result of in Utero Zidovudine Exposure

Abstract: Mitochondrial toxicity was examined in near‐term fetuses of pregnant Erythrocebus patas monkeys given human equivalent doses of 3′‐azido‐3′ deoxythymidine (AZT) during the second half of gestation. Pregnant monkeys were dosed daily with 10 or 40 mg AZT, equivalent to about 21% and 86% of the daily AZT dose (500 mg) given to HIV‐1‐positive pregnant women to prevent maternal‐fetal virus transmission. The fetal tissues examined include heart and skeletal muscle, which have high energy requirements, and placenta, which is less dependent on mitochondrial integrity. Slot blot quantitation of mitochondrial DNA (mtDNA) levels showed dose‐dependent depletion in heart, skeletal muscle, and placenta from AZT‐exposed fetuses compared to unexposed controls. Furthermore, mtDNA degradation, observed by Southern blot analysis, appeared more extensive in AZT‐exposed tissues compared to unexposed controls. Mitochondrial functional integrity, as determined by oxidative phosphorylation (OXPHOS) enzyme assays, was also examined in heart, skeletal muscle, and placenta. All three tissues showed strong dose‐related decreases in Complex I. In placenta, dose‐related increases for Complexes II and IV and a decrease for Complex III were observed. Dose‐related increases for Complexes II and IV observed in heart and skeletal muscle have been reported. 1 The increase in Complex IV (cytochrome c oxidase) activity in heart and skeletal muscle tissue from patas fetuses exposed to 40 mg AZT/day has been confirmed here by histochemical staining. Overall, data demonstrate that mitochondrial toxicity, evidenced by depletion in mtDNA and OXPHOS enzyme abnormalities, is manifested similarly in heart, skeletal muscle, and placenta of AZT‐exposed monkey fetuses. It is therefore possible that the placenta, which is a readily accessible tissue, might be an indicator of potential mitochondrial toxicity in human pregnancies involving nucleoside analog drug exposure.