Effects of Zidovudine Treatment on Heart mRNA Expression and Mitochondrial DNA Copy Number Associated with Alterations in Deoxynucleoside Triphosphate Composition in a Neonatal Rat Model

Abstract

The prevention of mother-to-child transmission (MTCT) of HIV is a crucial component in HIV therapy. Nucleoside reverse transcriptase inhibitors (NRTIs), primarily 3'-azido-3'-thymidine (AZT [zidovudine]), have been used to treat both mothers and neonates. While AZT is being replaced with less toxic drugs in treating mothers in MTCT prevention, it is still commonly used to treat neonates. Problems related to mitochondrial toxicity and potential mutagenesis associated with AZT treatment have been reported in treated cohorts. Yet little is known concerning the metabolism and potential toxicity of AZT on embryonic and neonatal tissues, especially considering that the enzymes of nucleoside metabolism change dramatically as many tissues convert from hyperplastic to hypertrophic growth during this period. AZT is known to inhibit thymidine phosphorylation and potentially alter deoxynucleoside triphosphate (dNTP) pools in adults. This study examines the effects of AZT on dNTP pools, mRNA expression of deoxynucleoside/deoxynucleotide metabolic enzymes, and mitochondrial DNA levels in a neonatal rat model. Results show that AZT treatment dramatically altered dNTP pools in the first 7 days of life after birth, which normalized to age-matched controls in the second and third weeks. Additionally, AZT treatment dramatically increased the mRNA levels of many enzymes involved in deoxynucleotide synthesis and mitochondrial biogenesis during the first week of life, which normalized to age-matched controls by the third week. These results were correlated with depletion of mitochondrial DNA noted in the second week. Taken together, results demonstrated that AZT treatment has a powerful effect on the deoxynucleotide synthesis pathways that may be associated with toxicity and mutagenesis.

FIG 1

Effects of AZT treatment on mRNA levels and RPL4 gene. mRNA and cDNA were obtained from hearts of age-matched controls and AZT-treated embryonic (day −1) and neonatal (postgestation days 1, 3, 7, 14, and 21) pups as described in Materials and Methods. (A) mRNA was quantified and expressed as OD₂₆₀ value per milligram of heart tissue. (B) Effect of AZT on housekeeping (RpL4) gene mRNA expression level. Results are expressed as described in Materials and Methods as fold changes from cycle time of adult control Dguok for both treated and aged-matched control samples. All data points represent means and SEM from three independent determinations from three different rat heart isolates. Student's t test was performed to analyze the significance between the control and the treated groups at each time point. *, P < 0.05; **, P < 0.01.

FIG 2

Total dNTP pool in embryonic and neonatal pup heart tissue. dNTP pools from hearts of age-matched controls and AZT-treated (100 mg/kg body weight) embryonic (day −1) and neonatal (postgestation days 1, 3, 7, 14, and 21) pups were quantified using a modified template-driven DNA polymerase assay described in Materials and Methods. The sums of all four dNTPs from each heart tissue were expressed as picomoles per milligram of tissue. All data points represent means and SEM from three independent determinations from three different rat heart isolates. Student's t test was performed to analyze the significance between the control and the treated groups at each time point. *, P < 0.05; **, P < 0.01.

FIG 3

Effects of AZT treatment on total dNTP pool composition and asymmetry in embryonic and neonatal pup heart tissue. dNTP pools from age-matched controls and AZT-treated rat heart tissues were prepared and quantified as described in Materials and Methods and the legend to Fig. 2. The composition of each dNTP is expressed as a percentage of the total dNTP of the heart at each time point. Totals represent the absolute amount of total dNTP expressed as picomoles per milligram of tissue. All data points represent means and SEM from three independent determinations from three different rat heart isolates. Student's t test was performed to analyze the significance of the percent composition of each dNTP between the control and the treated groups at each time point. *, P < 0.05; **, P < 0.01.

FIG 4

Effects of AZT treatment on individual dNTP concentrations in embryonic and neonatal pup heart tissue. dNTPs from age-matched controls and AZT-treated rat heart tissues were extracted and quantified as described in Materials and Methods and the legend to Fig. 2. The concentration of each dNTP is expressed in picomoles per milligram of tissue. (A) TTP; (B) dCTP; (C) dGTP; (D) dATP. All data points represent means and SEM from three independent determinations from three different rat heart isolates. Student's t test was performed to analyze the significance between the control and the treated groups at each time point. *, P < 0.05; **, P < 0.01.

FIG 5

mRNA expression in embryonic and neonatal rat heart tissue. Thirty nanograms of heart cDNA obtained from age-matched controls and AZT-treated embryonic (day −1) and neonatal (postgestation days 1, 3, 7, 14, and 21) pups was used in real-time PCR as described in Materials and Methods. mRNA expression levels were expressed as fold changes as described in the legend to Fig. 1. Primer sequences used in this study are shown in Table 1. All data points represent means and SEM from three independent determinations from three different rat heart isolates. Student's t test was performed to analyze the significance between the control and the treated groups at each time point. *, P < 0.05; **, P < 0.01.

FIG 6

Mitochondrial DNA copy numbers in embryonic and neonatal rat heart tissue. Genomic and mitochondrial DNAs from age-matched controls and AZT-treated rat hearts were isolated as described in Materials and Methods. Expression of mitochondrial DNA copy number was quantified by subtracting the mitochondrial gene (COX1 gene) cycle time from that of the nuclear gene (RpL4 gene) and expressed as mtDNA copy number per diploid nuclear genome. All data represent means and SEM from three independent determinations from three different rat heart isolates. Student's t test was performed to analyze the significance between the control and the treated groups at each time point. *, P < 0.05.