RESUMO
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.
Assuntos
Antimutagênicos/farmacologia , Benzo(a)pireno/metabolismo , Clorofilídeos/farmacologia , Citocromo P-450 CYP1A1/genética , Sistema Enzimático do Citocromo P-450/genética , Adutos de DNA/metabolismo , Glândulas Mamárias Humanas/efeitos dos fármacos , Hidrocarboneto de Aril Hidroxilases , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1 , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Medições Luminescentes , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/enzimologia , Glândulas Mamárias Humanas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Antiretroviral nucleoside reverse transcriptase inhibitors (NRTIs), given to human immunodeficiency virus-1-infected pregnant women to prevent vertical viral transmission, have caused mitochondrial dysfunction in some human infants. Here, we examined mitochondrial integrity in skeletal muscle from offspring of pregnant retroviral-free Erythrocebus patas dams administered human-equivalent NRTI doses for the last 10 weeks of gestation or for 10 weeks of gestation and 6 weeks after birth. Exposures included no drug, Zidovudine (AZT), Lamivudine (3TC), AZT/3TC, AZT/Didanosine (ddI), and Stavudine (d4T)/3TC. Offspring were examined at birth (n=3 per group) and 1 year (n=4 per group, not including 3TC alone). Circulating levels of creatine kinase were elevated at 1 year in the d4T/3TC-exposed group. Measurement of oxidative phosphorylation enzyme activities (complexes I, II, and IV) revealed minimal NRTI-induced changes at birth and at 1 year. Histochemistry for complex IV activity showed abnormal staining with activity depletion at birth and 1 year in groups exposed to AZT alone and to the 2-NRTI combinations. Electron microscopy of skeletal muscle at birth and 1 year of age showed mild to severe mitochondrial damage in all the NRTI-exposed groups, with 3TC inducing mild damage and the 2-NRTI combinations inducing extensive damage. At birth, mitochondrial DNA (mtDNA) was depleted by approximately 50% in groups exposed to AZT alone and the 2-NRTI combinations. At 1 year, the mtDNA levels had increased but remained significantly below normal. Therefore, skeletal muscle mitochondrial compromise occurs at birth and persists at 1 year of age (46 weeks after the last NRTI exposure) in perinatally exposed young monkeys, suggesting that similar events may occur in NRTI-exposed human infants.
Assuntos
Fármacos Anti-HIV/toxicidade , Exposição Materna/efeitos adversos , Mitocôndrias Musculares/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Inibidores da Transcriptase Reversa/toxicidade , Animais , Animais Recém-Nascidos , Creatina Quinase/metabolismo , DNA Mitocondrial/análise , DNA Mitocondrial/efeitos dos fármacos , Quimioterapia Combinada , Erythrocebus patas , Feminino , Idade Gestacional , Histocitoquímica , Microscopia Eletrônica de Transmissão , Mitocôndrias Musculares/genética , Mitocôndrias Musculares/ultraestrutura , Complexos Multienzimáticos/metabolismo , Músculo Esquelético/enzimologia , Músculo Esquelético/ultraestrutura , GravidezRESUMO
Effective reduction in maternal-fetal human immunodeficiency virus-1 (HIV-1) transmission has been achieved by administration of nucleoside reverse transcriptase inhibitors (NRTIs) during pregnancy, and although most exposed children are clinically normal at birth, mitochondrial dysfunction has been reported. To examine mitochondrial integrity on a molecular level, we evaluated mitochondrial morphology by electron microscopy (EM) and mitochondrial DNA (mtDNA) quantity in umbilical cords and cord blood from NRTI-exposed and unexposed human and monkey newborns. Human subjects included infants born to HIV-1-infected mothers who received Combivir (Zidovudine [AZT] plus Lamivudine [3TC]) (n = 9) or AZT plus Didanosine [ddI] (n = 2) during pregnancy, and infants born to HIV-1-uninfected mothers (n = 7). NRTI-exposed Erythrocebus patas monkey dams (n = 3 per treatment group) were given human-equivalent dosing regimens containing 3TC, AZT/3TC, AZT/ddI, or Stavudine (d4T)/3TC during gestation. Four infants born to unexposed patas dams served as controls. Mitochondria in umbilical cord endothelial cells from NRTI-exposed monkey and human infants showed substantial abnormal pathology by EM, the extent of which was quantified from coded photomicrographs and shown to be different (P < 0.05) from the unexposed monkey and human newborns. Significant (P < 0.05) mtDNA depletion was found in umbilical cords from both human and monkey NRTI-exposed infants and in human, but not in monkey, cord blood leukocytes. For umbilical cords, an increase in mitochondrial morphological damage correlated with reduction in mtDNA quantity in fetal monkeys (r = 0.94). The treatment-induced mitochondrial compromise in infant monkeys ranked as follows: d4T/3TC > AZT/ddI > AZT/3TC > 3TC. The study demonstrates that transplacental NRTI exposures induce similar mitochondrial damage in cord blood and umbilical cords taken from retroviral-uninfected monkey infants and from human infants born to HIV-1-infected women.
Assuntos
Fármacos Anti-HIV/efeitos adversos , Modelos Animais de Doenças , Mitocôndrias/efeitos dos fármacos , Inibidores da Transcriptase Reversa/efeitos adversos , Animais , Animais Recém-Nascidos , Fármacos Anti-HIV/administração & dosagem , Fármacos Anti-HIV/toxicidade , Ensaios Clínicos como Assunto , DNA Mitocondrial/análise , Didanosina/efeitos adversos , Combinação de Medicamentos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Células Endoteliais/ultraestrutura , Erythrocebus patas , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/prevenção & controle , Humanos , Recém-Nascido , Lamivudina/efeitos adversos , Troca Materno-Fetal , Mitocôndrias/patologia , Mitocôndrias/ultraestrutura , Gravidez , Complicações Infecciosas na Gravidez/tratamento farmacológico , Complicações Infecciosas na Gravidez/prevenção & controle , Estudos Retrospectivos , Inibidores da Transcriptase Reversa/administração & dosagem , Inibidores da Transcriptase Reversa/toxicidade , Cordão Umbilical/metabolismo , Cordão Umbilical/ultraestrutura , Zidovudina/efeitos adversosRESUMO
Hearts from 1-yr-old Erythrocebus patas monkeys were examined after in utero and 6-wk-postbirth exposure to antiretroviral nucleoside reverse transcriptase inhibitors (NRTIs). Protocols were modeled on those given to human immunodeficiency virus (HIV)-1-infected pregnant women. NRTIs were administered daily to the dams for the last 20% or 50% of gestation, and to the infants for 6 wk after birth. Exposures included: no drug (n = 4); Zidovudine, 3'-azido-3'-deoxythymidine (AZT; n = 4); AZT/Lamivudine, (-)-beta-L-2', 3'-Dideoxy-3'-thiacytidine (Epivir, 3TC) (n = 4); AZT/Didanosine (Videx, ddI) (n = 4); and Stavudine (Zerit, d4T)/3TC (n = 4). Echocardiograms and clinical chemistry showed no drug-related changes, but the d4T/3TC-exposed fetuses at 6 and 12 mo had increased white cell counts (p < 0.05). At 1 yr of age, oxidative phosphorylation (OXPHOS) enzyme activities were similar in heart mitochondria from all groups. Mitochondrial pathology, that included clones of damaged mitochondria (p < 0.05), was found in hearts of all 1-yr drug-exposed infants. Levels of mtDNA were elevated (p < 0.05) in hearts of all NRTI-exposed monkeys in the following order: control < d4T/3TC < AZT < AZT/3TC < AZT/ddI. The clinical status of NRTI-exposed infants, as evidenced by behavior, clinical chemistry, OXPHOS activity and echocardiogram, was normal. However, extensive mitochondrial damage with clusters of similar-appearing damaged heart mitochondria observed by electron microscopy, and an increase in mtDNA quantity, that persisted at 1 yr of age, suggest the potential for cardiotoxicity later in life.