Warnings from Experiments with Animals

“mice were treated with AZT for 35 days (10 mg/kg/day) in drinking water. Animals treated with antioxidant vitamins were fed the same diet as controls but supplemented with vitamins C (ascorbic acid, 10 g/ kg diet) and E (alpha-dl-tocopherol, 0.6 g/kg diet) for 65 days before sacrifice…Cardiac mitochondrial DNA (mtDNA) of mice treated with AZT had over 120% more oxo-dG (8-oxo-7,8-dihydro-2'-deoxyguanosine, which is a biomarker of oxidative damage to DNA) in their mitochondrial DNA than untreated controls. AZT treatment also caused an increase in mitochondrial lipid peroxidation and an oxidation of mitochondrial glutathione…The oxidative effects of AZT are probably due to an increase in production of reactive oxygen species by mitochondria of AZT-treated animals, raising the possibility that oxidative stress may play an important role in the cardiotoxicity of AZT.”

“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 [AZT]) 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.”

“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 six-fold 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.”

“CD-1 mice exposed prenatally to 12.5 and 25.0 mg of AZT...had statistically significant increases in numbers of liver, lung and female reproductive tract tumors. These observations have been extended to offspring at 2 years of age...there was a 2- to 3-fold increase in the incidence (from 20% in controls to 55-60% in AZT groups) and multiplicities of lung tumors in AZT-exposed mice. The incidence of hepatocellular adenomas in the female mice exposed to prenatal AZT increase from 0 in the control group to 20% in the high dose AZT group, and hepatocellular carcinomas metastasizing to lungs were observed only in AZT-treated mice. Prenatal administration of AZT also increased the incidence of neoplasms of reproductive tract, female mammary gland epithelium and squamous cell epithelium of forestomach. AZT...significantly reduced the incidence of hematopoietic tumors”

“The AZT animals [Macaques given AZT during pregnancy] developed an asymptomatic macrocytic anemia, but hematologic parameters returned to normal when AZT was discontinued. Total leukocyte count decreased during pregnancy and was further affected by AZT administration. AZT-exposed infants were mildly anemic at birth. AZT caused deficits in growth, rooting and snouting reflexes, and the ability to fixate and follow near stimuli visually”

“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...AZT is genotoxic in fetal mice and monkeys and is a moderately strong transplacental carcinogen in mice examined at 1 year of age”

“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.”

“…in adult mice, lifetime AZT administration induces vaginal tumors at a 10-20% incidence...In newborn monkeys and mice, AZT was incorporated into DNA of many fetal tissues...AZT appears to be a moderately-strong transplacental carcinogen [i.e. it crosses the placenta and may cause cancer in the fetus]”

“Significant dose-related increases in the percentage [of] micro-nucleated erthyrocytes [red blood cells] in the peripheral blood were observed in mice treated with ZDV [AZT]…Bone marrow micronucleus frequencies were also elevated in mice and rats treated with ZDV at 500 mg/kg/day for 4 (mice only) and 7 days…seven late-appearing vaginal epithelial cell tumors were seen in mice given 40 mg/kg/day…Spontaneous tumors of the vaginal epithelium are rare in rodents, and reports of chemically induced vaginal tumors are also uncommon. Of 222 chemicals evaluated for carcinogenicity in rats and mice in the National Toxicology Program, none induced tumors in the vagina…In the CRC 'Handbook of Identified Carcinogens and Noncarcinogens' (1982), 356 of the 811 compounds listed were shown to be carcinogenic in rodents. Of the 356 positive chemicals, only 3 caused vaginal neoplasms, DES [diethylstilbestrol, associated with major genital deformities in humans], estradiol 3-benzoate, and 17 Beta-estradiol”

“Hemoglobin dropped significantly in the AZT-treated animals [Macaques] after treatment began and remained low until the end of the study...Postnatal weight increase was significantly lower in AZT-exposed infants...Infant hematocrits taken at time of birth were lower in the AZT-exposed group...AZT-exposed infants took three times as many sessions as controls to meet criterion on Black-White Learning, a simple discrimination task...It took significantly more matings to achieve the six AZT pregnancies than the six control pregnancies”

“we have found positive correlations between the dose of AZT administered to female CD-1 mice, the incorporation of AZT into vaginal DNA, the hyperproliferation of the vaginal epithelial basal layer, and the aberrant expression of alpha-6 integrin toward the epithelial suprabasal strata of the vagina, a target organ for carcinogenesis in mice. These results suggest that there is an ordered progression of abnormal events leading to tumorigenesis in vaginal epithelial tissues.”

“It previously has been demonstrated that zidovudine (AZT) is lethal to early murine [mouse] embryos. The effect of the drug on pre- and postimplantation embryos was examined to delineate the timing of this toxicity and to investigate its possible mechanisms. Embryos exposed in the whole mouse during preblastocyst development were unable to proceed beyond the blastocyst stage [i.e. failed to implant in the uterine wall]. Similarly, when two-cell embryos harvested from unexposed females were exposed to low-concentration (1 microMole) AZT in vitro over 24 h, development beyond the blastocyst stage was inhibited. In contrast, drug exposure during in vitro blastocyst and postblastocyst development resulted in little or no morphologic toxicity. Further investigation revealed that preblastocyst AZT exposure resulted in the development of blastocysts with significantly lower cell numbers than control embryos. While embryonic exposure to AZT at the blastocyst and postblastocyst stages also resulted in retarded cell division, the effects were milder than those recorded after preblastocyst exposure. These data demonstrate that the critical period of AZT toxicity toward murine embryos is between ovulation and implantation and indicate that AZT directly suppresses cell division in the preimplantation embryo.”

“6 adult…rats were treated intraperitoneally either with AZT or with normal saline twice daily for 3 months. The AZT was used at doses equivalent to the high therapeutic dose administered daily to patients with AIDS…The AZT-treated animals lost 10% of the original weight by the end of the 3rd month…the animals appeared sluggish and at times drowsy. Serum creatine kinase was dramatically increased in the AZT-treated animals…Serum lactate was increased in the treated animals…Slight anemia with hematocrit as low as 35% (normal 40 to 49%), thrombocytopenia with platelet count between 119 to 508/cubic-mm (normal 610 to 1,610) and lymphocytopenia with white blood cell count ranging from 2,900 to 4,200 (normal 9,400 to 14,900) were noted in the AZT-treated group. The animal that received the highest dose of AZT developed hyperglycemia with blood sugar up to 500 mg/ml (upper limit of normal 120 mg/ml). Muscle biopsies from the AZT-treated rats, but not the controls, showed subsarcolemmal accumulations…suggesting early formation of ‘ragged red’ fibers. With electron microscopy, there was clear evidence of abnormal mitochondria”

“Striking differences between the AZT-treated hearts and control rat hearts were found ultrastructurally. Myocardial [heart muscle] samples from the AZT-treated group revealed widespread cardiac myocyte mitochondrial disruption and fragmentation of cristae. Some mitochondrial cristae appeared focally dissolved and poorly organized…Other zones showed mitochondrial cristae that were clumped and focally swollen. Mitochondrial findings were consistently present in multiple samples of myocytes from AZT-treated rat hearts and conspicuously absent from samples of hearts of untreated rats.”

“Mice receiving AZT during gestation yielded fewer fetuses…and greater numbers of resorptions [fetal death]…Exposure to AZT was highly correlated with failure to develop to the blastocyst stage…These data indicate that AZT has a direct toxic effect on the developing mouse embryo.”

“The most consistent hematologic effect from treatment with AZT [in mice] was a poorly regenerative, macrocytic anemia”

“[in mice] AZT had a profound effect on the number of erythrocytes [mature red blood cells] and a small effect on the number of leukocytes [white blood cells]…anemia was seen in all the mice tested at 1,000 mg/kg per day”

“Male and female cynomolgus monkeys were given zidovudine [AZT], 35 to 300 mg/kg per day orally, in studies of 3 and 6 months’ duration…the only treatment-related alteration noted was a reversible, mild to moderate, dose-related, macocytic anemia…In the 6-month study, bone marrow cytology…revealed a retardation in the maturation of all cell lines, with the ertythroid elements [red blood cells] being affected to the greatest degree…Dams [pregnant female rabbits] given 500 mg/kg per day gained less weight during the dosing period, developed anemia, and showed an increased incidence of late fetal resorptions. No evidence of teratogenicity [birth defects] was seen, even though it was shown that zidovudine crossed the placenta…Zidovudine was also studied for its ability to morphologically transform cultured BALB/c-3T3 mouse cells and was found to be positive at concentrations of 0.5 micrograms/ml and higher…From the Department of Toxicology and Experimental Pathology. Burroughs Wellcom Co. [the manufacturer of AZT]”