Transplacental effects of 3'-azido-2',3'-dideoxythymidine (AZT): tumorigenicity in mice and genotoxicity in mice and monkeys.

BACKGROUND: When given during pregnancy, the drug 3'-azido-2',3'-dideoxythymidine (AZT) substantially reduces maternal-fetal transmission of human immunodeficiency virus type 1 (HIV-1). However, AZT has been shown to be carcinogenic in adult mice after lifetime oral administration. In this study, we assessed the transplacental tumorigenic and genotoxic effects of AZT in the offspring of CD-1 mice and Erythrocebus patas monkeys given AZT orally during pregnancy. METHODS: Pregnant mice were given daily doses of either 12.5 or 25.0 mg AZT on days 12 through 18 of gestation (last 37% of gestation period). Pregnant monkeys were given a daily dose of 10.0 mg AZT 5 days a week for the last 9.5-10 weeks of gestation (final 41%-43% of gestation period). AZT incorporation into nuclear and mitochondrial DNA and the length of chromosomal end (telomere) DNA were examined in multiple tissues of newborn mice and fetal monkeys. Additional mice were followed from birth and received no further treatment until subjected to necropsy and complete pathologic examination at 1 year of age. An anti-AZT radioimmunoassay was used to monitor AZT incorporation into DNA. RESULTS: At 1 year of age, the offspring of AZT-treated mice exhibited statistically significant, dose-dependent increases in tumor incidence and tumor multiplicity in the lungs, liver, and female reproductive organs. AZT incorporation into nuclear and mitochondrial DNA was detected in multiple organs of transplacentally exposed mice and monkeys. Shorter chromosomal telomeres were detected in liver and brain tissues from most AZT-exposed newborn mice but not in tissues from fetal monkeys. CONCLUSIONS: AZT is genotoxic in fetal mice and monkeys and is a moderately strong transplacental carcinogen in mice examined at 1 year of age. Careful long-term follow-up of AZT-exposed children would seem to be appropriate.

Transplacental cisplatin exposure induces persistent fetal mitochondrial and genomic DNA damage in patas monkeys.

A previous attempt to model transplacental cisplatin exposure and genotoxicity employed several pregnant Erythrocebus patas monkeys; most of the animals were exposed near the end of gestation and cisplatin-DNA adduct analyses included only genomic DNA. Here, both genomic and mitochondrial DNA adduct formation have been determined in fetuses from two pregnant monkeys exposed at the end of the second trimester of gestation. Multiple fetal tissues were obtained after doses of 0.315 mg cisplatin/kg body weight (5.3 mg/m2 total) on days 101 and 106 of gestation. Cesarean sections were performed 24 h after exposure and 27 d after exposure. Cisplatin genomic (g)-DNA adducts were observed in fetal adrenal, brain, heart, kidney, liver, skin, spleen, and thymus. When placentas from the two animals were divided into four concentric regions at increasing distances from the umbilical cord, and g-DNA was assayed, cisplatin DNA adduct levels were similar in all four regions. Mitochondrial (mt)-DNA adducts were higher than g-DNA adducts in maternal liver and fetal liver, brain and kidney, suggesting that the mitochondria may constitute a particular target for cisplatin genotoxicity. The study demonstrates significant fetal genotoxicity in g-DNA and mt-DNA of patas monkeys exposed to cisplatin in utero, suggesting that similarly exposed human fetuses may also sustain drug-induced DNA damage.

N-nitrosodimethylamine-derived O(6)-methylguanine in DNA of monkey gastrointestinal and urogenital organs and enhancement by ethanol.

N-nitrosodimethylamine (NDMA) is a human cancer initiator suspect. Ethanol, a cancer risk factor, may synergize with nitrosamines by suppressing hepatic clearance, to increase internal exposure. A limitation to these hypotheses is lack of activation of NDMA by many rodent tissues. However, systemtic primate studies are lacking. Patas monkeys were utilized to investigate NDMA activation by primate tissues in vivo, generating the promutagenic DNA lesion 0(6)-methylguanine (0(6)-meG). Adult monkeys received 0. 1 mg/kg NDMA by gavage, in some cases preceded by ethanol. Four hours after NDMA only, 0(6)-meG was detected in DNA from all tissues. Levels were highest in gastric mucosa and liver and were only about 50% lower in DNA from white blood cells, esophagus, ovary, pancreas, urinary bladder and uterus. With ethanol co-exposure, amounts of 0(6)-meG increased at least 2-fold in all tissues except liver. The largest effect was in esophagus (17-fold increase), followed by ovary, large intestine, urinary bladder, spleen and cerebellum (9- to 13-fold increases), and uterus, cerebrum and brain stem (7- to 8-fold increases). Alkylguanine alkyltransferase activities varied over a 30-fold range and were highest in liver and stomach. Thus primate tissues, especially those of the gastrointestinal and urogenital organs, are sensitive targets for DNA adduct damage due to NDMA, and ethanol co-exposure leads to striking increases in adducts. Our data support epidemiology implicating nitrosamines in causation of cancers of stomach and other organs, and alcohol as enhancing internal exposure to nitrosamines.

DNA adducts in human and patas monkey maternal and fetal tissues induced by platinum drug chemotherapy.

Platinum-DNA adducts in placenta and blood from a woman exposed to 200 mg/m2 of cis-diamminedichloroplatinum(II) (cisplatin) and 300 mg/m2 diamminecyclobutanedicarboxylatoplatinum(II) (carboplatin) for ovarian cancer have been documented by cisplatin-DNA enzyme-linked immunosorbent assay (ELISA) and atomic absorbance spectrometry (AAS). A patas monkey model was used to investigate transplacentally induced cisplatin-DNA damage in fetal tissues. During the last trimester of gestation, 5 patas monkeys were given multiple doses of cisplatin to mimic human ovarian cancer treatment. In spite of careful choice of dose and treatment conditions, cumulative toxicity occurred in monkeys given doses comparable on a mg/m2 basis to those received by the human. A total dose of 12 mg/m2 (0.625 mg/kg body weight), given in the last trimester, supported fetal viability, and multiple tissues, taken by cesarean section, were examined in the fetal monkeys. By cisplatin-DNA ELISA and AAS, maternal tissues from the monkey receiving the highest dose contained approximately twice as much DNA damage as the fetal tissues. A similar relationship was observed when we compared DNA adduct formation in fetal liver and biopsies of liver taken from the monkey dams at cesarean delivery. In all of the monkey pairs studied there were very significant levels of DNA damage in the placenta, and high adduct levels in brains of fetuses that survived treatment. Thus, cisplatin does cross the placenta in the patas monkey. These observations imply that the human fetus, for which the total maternal dose was approximately 5.4 mg platinum drug/kg body weight, may also have sustained some DNA damage.

Persistence, gestation stage-dependent formation and interrelationship of benzo[a]pyrene-induced DNA adducts in mothers, placentae and fetuses of Erythrocebus patas monkeys.

Since DNA adducts have been detected in the placentae of pregnant women who smoke cigarettes, the importance of these adducts as biomarkers of fetal exposure and risk has been evaluated using a non-human primate as a model. Pregnant Erythrocebus patas monkeys on days 50, 100 or 150 of gestation (term = 160 +/- 5 days) were treated once with 5-50 mg/kg benzo[a]pyrene (B[a]P), p.o. Fetuses were removed by Cesarean section 1-50 days after treatment and analyzed for DNA adducts by the nuclease P1 version of the 32P-postlabeling method. B[a]P induced high levels of DNA adducts in all fetal organs, placentae and maternal livers in all three trimesters of gestation. DNA adduct levels were higher in mid-gestation compared to early and late gestation. The major adduct detected was 10 beta-(deoxyguanosin)-N2-yl-7 beta,8 alpha,9 alpha-trihydroxy-7,8,9,10- tetrahydro-B[a]P. The adduct levels in fetal tissues increased with B[a]P dose, but at a much lower rate than in placentae or maternal livers. Preference in binding to DNA of various fetal organs was more apparent in early gestation compared to late gestation and at lower doses compared to higher doses. During early gestation and at low doses, B[a]P produced a similar level of DNA adducts in fetal lung, fetal liver, maternal liver and placenta. Individual fetal organ adduct levels correlated significantly with placental adduct levels, indicating placental and/or maternal contribution to genotoxic injuries in fetuses. However, the slopes of linear regression lines of correlation analyses varied among organs and among gestation stages at treatment, indicating fetal contribution to its own genotoxic injuries. DNA adduct levels in fetal skin were the lowest of all fetal organs tested and less affected by gestational stages at time of treatment. In contrast, DNA adduct levels in fetal liver exhibited distinct gestation stage specificity with higher adduct levels attained during mid-gestation compared to other stages of gestation. Adduct levels decreased at a much faster rate during the first 10-15 days compared to 15-50 days after B[a]P treatment. However, 10% of DNA adducts persisted 50 days after treatment in all organs studied. Together, the results suggest that placental adduction accurately indicates fetal exposure. Toxicokinetics of B[a]P and its metabolites as well as maternal, placental and fetal competence in activation and deactivation of B[a]P may be critical determinants in overall fetal risk to genetic damage. Importantly, maximal sensitivity to transplacental DNA damage may be during mid-gestation.

Reduced blood clearance and increased urinary excretion of N-nitrosodimethylamine in patas monkeys exposed to ethanol or isopropyl alcohol.

Low concentrations of N-nitrosodimethylamine are metabolized in rodent and human liver by cytochrome P450IIE1, an activity competitively inhibitable by ethanol. In rodents coadministration of ethanol with N-nitrosodimethylamine results in increased tumorigenicity in extrahepatic organs, probably as a result of reduced hepatic clearance. To test this concept in a primate, the effects of ethanol cotreatment on the pharmacokinetics of N-nitrosodimethylamine were measured in male patas monkeys. Ethanol, 1.2 g/kg given p.o. before i.v. N-nitrosodimethylamine (1 mg/kg) or concurrently with an intragastric dose resulted in a 10-50-fold increase in the area under the blood concentration versus time curves and a 4-13-fold increase in mean residence times for N-nitrosodimethylamine. Isopropyl alcohol, 3.2 g/kg 24 h before N-nitrosodimethylamine, also increased these parameters 7-10-fold; this effect was associated with persistence of isopropyl alcohol and its metabolic product acetone, both IIE1 inhibitors, in the blood. While no N-nitrosodimethylamine was detected in expired air, trace amounts were found in urine. Ethanol and isopropyl alcohol pretreatment increased the maximum urinary N-nitrosodimethylamine concentration 15-50-fold and the percentage of the dose excreted in the urine by 100-800-fold. Thus ethanol and isopropyl alcohol greatly increase systemic exposure of extrahepatic organs to N-nitrosodimethylamine in a primate.

A technique for liver biopsy in Pekin ducks.

The duck hepatitis B virus (DHBV), a member of the hepadna-virus group, has become a useful animal model for exploring important aspects in this family of viruses such as viral replication, course of infection, and the response to antiviral therapy. In chronically DHBV infected ducks, repeated analyses of liver tissue are important in defining the degree of viral replication and liver injury. We describe a technique for repeated liver biopsy using a Keyes skin punch biopsy. This technique provided sufficient quantities of liver tissue for serial analyses with minimal hemorrhage in 18 Pekin ducks. This procedure offers a safe and reliable method of obtaining serial liver biopsies.