Progressive mitochondrial compromise in brains and livers of primates exposed in utero to nucleoside reverse transcriptase inhibitors (NRTIs).

Mitochondrial compromise has been documented in infants born to women infected with the human immunodeficiency virus (HIV-1) who received nucleoside reverse transcriptase inhibitor (NRTI) therapy during pregnancy. To model these human exposures, we examined mitochondrial integrity at birth and 1 year in brain cortex and liver from offspring of retroviral-free Erythrocebus patas dams-administered human-equivalent NRTI doses for the last half (10 weeks) of gestation. Additional infants, followed for 1 year, were given the same drugs as their mothers for the first 6 weeks of life. Exposures included: no drug, Zidovudine (AZT), Lamivudine (3TC), AZT/3TC, AZT/Didanosine (ddI), and Stavudine (d4T)/3TC. In brain and liver, oxidative phosphorylation (OXPHOS) enzyme activities (complexes I, II, and IV) showed minimal differences between unexposed and NRTI-exposed offspring at both times. Brain and liver mitochondria from most NRTI-exposed patas, both at birth and 1 year of age, contained significant (p < 0.05) morphological damage observed by electron microscopy (EM), based on scoring of coded photomicrographs. Brain and liver mitochondrial DNA (mtDNA) levels in NRTI-exposed patas were depleted significantly in the 3TC and d4T/3TC groups at birth and were depleted significantly (p < 0.05) at 1 year in all NRTI-exposed groups. In 1-year-old infants exposed in utero to NRTIs, mtDNA depletion was 28.8-51.8% in brain and 37.4-56.5% in liver. These investigations suggest that some NRTI-exposed human infants may sustain similar mitochondrial compromise in brain and liver and should be followed long term for cognitive integrity and liver function.

In utero exposure of female CD-1 mice to AZT and/or 3TC: II. Persistence of functional alterations in cardiac tissue.

To delineate temporal changes in the integrity and function of mitochondria/cardiomyocytes in hearts from mice exposed in utero to commonly used nucleoside analogs (NRTIs), CD-1 mice were exposed in utero to 80 mg AZT/kg, 40 mg 3TC/kg, 80 mg AZT/kg plus 40 mg 3TC/kg, or vehicle alone during days 12-18 of gestation and hearts from female mouse offspring were examined at 13 and 26 weeks postpartum. Alterations in cardiac mitochondrial DNA (mtDNA) content, oxidative phosphorylation (OXPHOS) enzyme activities, mtDNA mutations, and echocardiography of NRTI-exposed mice were assessed and compared with findings in vehicle-exposed control mice. A hybrid capture-chemiluminescence assay showed significant twofold increases in mtDNA levels in hearts from AZT- and AZT/3TC-exposed mice at 13 and 26 weeks postpartum, consistent with near doubling in mitochondrial numbers over time compared with vehicle-exposed mice. Echocardiographic measurements at 13 and 26 weeks postpartum indicated progressive thinning of the left ventricular posterior wall in NRTI-exposed mice, relative to controls, with differences becoming statistically significant by 26 weeks. Overall, progressive functional changes occurred in mouse mitochondria and cardiac tissue several months after in utero NRTI exposures; AZT and 3TC acted in concert to cause additive cardiotoxic effects of AZT/3TC compared with either drug alone.

Chlorophyllin significantly reduces benzo[a]pyrene-DNA adduct formation and alters cytochrome P450 1A1 and 1B1 expression and EROD activity in normal human mammary epithelial cells.

We hypothesized that chlorophyllin (CHLN) would reduce benzo[a]pyrene-DNA (BP-DNA) adduct levels. Using normal human mammary epithelial cells (NHMECs) exposed to 4 microM BP for 24 hr in the presence or absence of 5 microM CHLN, we measured BP-DNA adducts by chemiluminescence immunoassay (CIA). The protocol included the following experimental groups: BP alone, BP given simultaneously with CHLN (BP+CHLN) for 24 hr, CHLN given for 24 hr followed by BP for 24 hr (preCHLN, postBP), and CHLN given for 48 hr with BP added for the last 24 hr (preCHLN, postBP+CHLN). Incubation with CHLN decreased BPdG levels in all groups, with 87% inhibition in the preCHLN, postBP+CHLN group. To examine metabolic mechanisms, we monitored expression by Affymetrix microarray (U133A), and found BP-induced up-regulation of CYP1A1 and CYP1B1 expression, as well as up-regulation of groups of interferon-inducible, inflammation and signal transduction genes. Incubation of cells with CHLN and BP in any combination decreased expression of many of these genes. Using reverse transcription real time PCR (RT-PCR) the maximal inhibition of BP-induced gene expression, >85% for CYP1A1 and >70% for CYP1B1, was observed in the preCHLN, postBP+CHLN group. To explore the relationship between transcription and enzyme activity, the ethoxyresorufin-O-deethylase (EROD) assay was used to measure the combined CYP1A1 and CYP1B1 activities. BP exposure caused the EROD levels to double, when compared with the unexposed controls. The CHLN-exposed groups all showed EROD levels similar to the unexposed controls. Therefore, the addition of CHLN to BP-exposed cells reduced BPdG formation and CYP1A1 and CYP1B1 expression, but EROD activity was not significantly reduced.