Intermittent hypoxia regulates RNA polymerase II in hippocampus and prefrontal cortex.

Intermittent hypoxia (IH) is a major pathological factor in the development of neural deficits associated with sleep-disordered breathing. Here we demonstrate that IH lasting 2 or 30 days, but not sustained hypoxia (SH) of the same duration, was accompanied by several posttranslational modifications of the large subunit of RNA polymerase II, Rpb1, including hydroxylation of proline 1465, phosphorylation of serine 5 residues within the C-terminal domain, and nondegradative ubiquitylation. These modifications were found to occur in two regions of the brain, hippocampal region CA1 and the prefrontal cortex, but not in neocortex, brainstem and CA3 region of hippocampus. We also found that mice exposed to 14 or 30 days of IH, but not SH, demonstrated cognitive deficits in behavioral assays. Furthermore, by using the pheochromocytoma-derived PC12 cell line, we showed that, under in vitro IH conditions, induction of Rpb1 hydroxylation, phosphorylation, and ubiquitylation required that the von Hippel-Lindau protein be present. We hypothesize that the observed modifications of Rpb1 participate in regulating the expression of genes involved in mediating cognitive deficits evoked by chronic IH.

Chronic stavudine exposure induces hepatic mitochondrial toxicity in adult Erythrocebus patas monkeys.

OBJECTIVES: To understand the mitochondrial mechanisms underlying the lactic acidosis and hepatic steatosis seen in some HIV-1-infected individuals after long-term stavudine (d4T) exposure, we have explored mitochondrial integrity in adult monkeys (Erythrocebus patas) given a daily human equivalent dose of d4T for 78 days. STUDY DESIGN/METHODS: Three Erythrocebus patas (patas) monkeys were given 3 mg d4T orally twice daily (total 6 mg d4T), or approximately 1.2 mg d4T/kg body weight per day, for 78 days and compared with 3 unexposed animals. Blood taken from controls and from treated monkeys before and after drug exposure was subjected to a complete clinical chemistry profile. Liver and skeletal muscles were examined for oxidative phosphorylation enzyme specific activities, mitochondrial deoxyribonucleic acid (mtDNA) quantity by slot blot, and mtDNA integrity by Southern blot. RESULTS: Clinical chemistry assays demonstrated few significant differences; however, one d4T-exposed monkey had a serum lactate of 8.1 mmol/L after 78 days of oral d4T ingestion. Specific activities of oxidative phosphorylation Complexes I, II, and IV were significantly altered in both livers and skeletal muscles from the d4T-exposed animals, compared with the controls (p < or = 0.05). Significant depletion of mitochondrial DNA was observed in livers of drug-exposed monkeys, but not in skeletal muscle (p < or = 0.05). Further examination of liver DNA by Southern blot confirmed hepatic mtDNA depletion in drug exposed animals. CONCLUSIONS: The data suggest that direct examination of the liver may be required to elucidate clinical d4T-induced hepatotoxicity related to mitochondrial damage.

Genotoxic and functional consequences of transplacental zidovudine exposure in fetal monkey brain mitochondria.

Mitochondrial toxicity was assessed in the brains of developing Erythrocebus patas monkey fetuses exposed in utero to the nucleoside analogue drug zidovudine (3'-azido-3'deoxythymidine or AZT). Pregnant E. patas monkeys were given 0 (n = 5), 10 (n = 3), and 40 (n = 3) mg of AZT/day, equivalent to 21 and 86% of the human daily dose, for the last half (about 10 weeks) of gestation. Mitochondria were isolated from fetal cerebrum and cerebellum at birth and mitochondrial morphology was examined in these tissues by transmission electron microscopy (TEM). Oxidative phosphorylation (OXPHOS) enzyme specific activities were measured spectrophotometrically. Mitochondrial DNA (mtDNA) integrity and quantity were determined by Southern blot and slot blot analysis. In the cerebral mitochondria, reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase (complex I) specific activity decreased by 25% in monkeys treated with 40 mg of AZT/day compared with unexposed monkeys (p > or = .05). At the same AZT dose in the cerebral mitochondria, succinate dehydrogenase (complex II) and cytochrome c reductase (complex IV)-specific activities showed dose-dependent increases (p > or = .05), compared with those in controls. In the cerebellum, no difference was seen in mitochondrial OXPHOS enzyme activities between unexposed and exposed fetuses. Furthermore, TEM demonstrated no difference in mitochondrial morphology in frontal cerebrum or cerebellum from unexposed and exposed fetuses, and all fetuses had similar amounts of mtDNA in both tissues. Cerebral mtDNA degradation was noted in the highest AZT dosage group, whereas mtDNA from cerebellum was uneffected. Thus, in fetal patas monkeys given a human equivalent daily dose of AZT during the last half of pregnancy, mitochondria in the fetal cerebrum appear to sustain moderate damage, while the fetal cerebellum mitochondria were not effected.

Fetal mitochondrial heart and skeletal muscle damage in Erythrocebus patas monkeys exposed in utero to 3'-azido-3'-deoxythymidine.

3'-azido-3'-deoxythymidine (AZT) is given to pregnant women positive for the human immunodeficiency virus type 1 (HIV-1) to reduce maternal-fetal viral transmission. To explore fetal mitochondrial consequences of this exposure, pregnant Erythrocebus patas monkeys were given daily doses of 1.5 mg (21% of the human daily dose) and 6.0 mg (86% of the human daily dose) of AZT/kg body weight (bw), for the second half of gestation. At term, electron microscopy of fetal cardiac and skeletal muscle showed abnormal and disrupted sarcomeres with myofibrillar loss. Some abnormally shaped mitochondria with disrupted cristae were observed in skeletal muscle myocytes. Oxidative phosphorylation (OXPHOS) enzyme assays showed dose-dependent alterations. At the human-equivalent dose of AZT (6 mg of AZT/kg bw), there was an approximately 85% decrease in the specific activity of NADH dehydrogenase (complex I) and three- to sixfold increases in specific activities of succinate dehydrogenase (complex II) and cytochrome-c oxidase (complex IV). Furthermore, a dose-dependent depletion of mitochondrial DNA levels was observed in both tissues. The data demonstrate that transplacental AZT exposure causes cardiac and skeletal muscle mitochondrial myopathy in the patas monkey fetus.

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.

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.

Infiltrating angiolipoma of skeletal muscle. Transplacental induction in nonhuman primates by N-nitrosoethylurea.

BACKGROUND: In humans, relatively little is known on the association of prenatal exposure to cancer-causing agents and the development of specific tumors later in life as a consequence. Therefore, the effects on the offspring of carcinogen exposure during gestation and the development of tumors later in life were studied in nonhuman primates. EXPERIMENTAL DESIGN: Pregnancy was confirmed in Erythrocebus patas (patas) and Macaca mulatta (rhesus) by palpation at 27 to 40 days of gestation. Pregnant animals were treated once weekly intravenously from that time with N-nitrosoethylurea according to different dosing regimens for 6 to 19 weeks with 0.05 to 0.2 mmol/kg/injection. RESULTS: A common lesion developing in only the offspring of mothers treated early in pregnancy was identical with the human condition referred to as intramuscular angioma, hemangioma, or infiltrating angiolipoma of skeletal muscle. In the rhesus, one of 7 animals, and in the patas, 18 of 78 monkeys developed these processes (10 to 40% per group). The lesions typically arose within, infiltrated and displaced skeletal muscle. They occurred most commonly in the lower extremities, followed by the upper extremities and the head; they recurred in three cases of incomplete resection but did not metastasize. The tumors were seen mainly in young adults of both sexes (latency range: 4 to 76 months) and consisted of vessels of variable caliber, and to varying degrees, mature adipose and connective tissue, undifferentiated mesenchymal cells, and lymphoid cell aggregates. Ultrastructurally, the endothelium possessed numerous Weibel-Palade bodies and showed strong immunoreactivity for von Willebrand factor by immunohistochemistry and immunoelectron microscopy. CONCLUSIONS: The present investigation suggests a classification of these lesions as infiltrating angiolipoma of skeletal muscle originating from a pluripotent mesenchymal stem cell, caused by exposure to carcinogens during early pregnancy. The great clinical and morphologic similarity of this condition with that observed in humans suggests that it may likewise be caused by exposure to an agent during pregnancy.

Endocrine profile of pregnancy in the patas monkey (Erythrocebus patas).

Spontaneous toxemia occurs in approximately 6% of pregnancies in patas monkeys (Erythrocebus patas). To assess further the relevance of this animal as a model system for toxemia of pregnancy in humans, we characterized the endocrine profile of the patas pregnancy by analyzing weekly blood samples for estrone, estradiol, estriol, progesterone, aldosterone, and PRA throughout gestation. The profiles obtained bear striking resemblance to those described for human pregnancy, an unexpected finding since the patas monkey is not a higher hominoid. We conclude that the patas monkey may provide a model of toxemia relevant to study of the human disorder and is superior to other animal models which require surgical manipulation and are characterized by secondary hyperreninemia.

Reproductive performance of a laboratory breeding colony of patas monkeys (Erythrocebus patas).

Reproductive statistics were gathered over a 5½-year period on a colony of Erythrocebus patas. Pregnancies occurred throughout the year under laboratory conditions with a suggestion of a mating peak in the late fall and early winter. Menstrual cycles were monitored and found to average 30.6 days in length. Maximal vaginal cornification occured on day 15 of the cycle suggesting a midcycle ovulation. However, production of timed-mated pregnancies indicated ovulation occurred earlier and that breeding on days 10, 11, and 12 after menstruation was more likely to result in pregnancy. The gestation length was found to average 167.2 days in 142 harem-bred females and 167.5 days in 11 timed-mated pregnancies. Sixty-two percent of all pregnancies resulted in live births; 28% of the conceptions terminated with in-utero death of the fetus. Stillborn infants were delivered in 9% of the pregnancies. Infant mortality during the first 6 months of life was 10.2%. Females raised in the colony conceived their first offspring at approximately 3 years of age and males were able to sire infants at 3 years and 8 months.