Evaluation of the Genotoxicity of Antiretroviral Drugs on Allium Cepa and Male Fertility in Two Generations of Mice
A Thesis Submitted to the School of Postgraduate Studies, University of Lagos
TRACT The potential of the antiretroviral drugs zidovudine and nevirapine to induce genotoxicity was evaluated using the Allium cepa and the mice models. Four doses per antiretroviral were used in the Allium cepa chromosomal aberration assay while three doses per antiretroviral were used for the mouse study. The Allium cepa study consisted of the 96hr root growth inhibition, the 48hr recovery and the 48hr chromosomal aberration phases to determine the EC50; ascertain if root growth inhibition is reversible, and to observe chromosomal aberrations during mitosis respectively. In mice, the drugs were administered for the 56 days duration of spermatogenesis. Sperm counts, morphology anomalies, DNA content and quality (DNA fragmentation and nicks) were assessed in the parental (at mid- and full-term) and F1 (at full-term) generations of mice. Dominant lethal assays were done and the birth statistics (gestation, litter size and birth weight) were compared to relate the effects on fertility. Exposure to zidovudine and nevirapine altered the mitotic phase distribution profile in Allium cepa with the dividing cells being mostly at telophase and prophase. Sticky chromosomes (23-62%) was the predominant aberration seen, followed by anaphase chromosomal bridges. In parental generation of mice, significant oligospermia (2.9 – 5.4 x 106/ml), increased anomalies in sperm morphology (2.1 – 10.6%) and elevated sperm DNA quantity (80.9 – 121.3 μg/ml) were observed. Sperm counts for the treatment groups were generally just above half the negative control value while almost five out of six treatment groups had their sperm anomaly indices at least twice that of the negative control. Fertility was increased about 3 times when both parents were ARV-treated contrasting with reduced fertility when only one parent was on ARVs. Shorter gestation periods (18.8 – 25.0 days) were recorded for all treatment groups compared to the unexposed controls (26 ± 1.4 days). Early foetal loss was evident in the nevirapine groups from the dominant lethal assays. Across the parental and the F1 generation, significant reductions in testis weight (pvalues = 0.001, 0.006 and 0.011) and severe oligospermia (0.3 – 3.9 x 106/ml) were observed and reduced sperm DNA quantity (except for one group; 19.3 – 21.5 μg/ml) was recorded. In the F1 generation, sperm DNA nicks were reduced 3-5 folds and fertility was reduced by 94.4%. In this study, Allium cepa demonstrated some earlier reported genotoxic effects of the ARVs administered, indicating it can be used to investigate their genotoxicity. Fertility in mice was enhanced when both parent received ARVs prior to mating. Mice that received zidovudine and nevirapine treatment prior to mating, whose parents were as well exposed to these drugs, exhibited highly reduced fertility. The reduced sperm counts, high abnormal sperm morphology and altered sperm DNA content amongst other effects observed in this study are attributable to the administration of zidovudine and nevirapine not HIV infection. It is recommended that when evaluating sperm DNA quality, the DNA nicks (via alkaline annealing method) had better correlation with reproductive outcomes and may thus be preferred.