Alkyltransferase-mediated toxicity of bis-electrophiles in mammalian cells.

The primary function of O(6)-alkylguanine-DNA alkyltransferase (AGT) is to maintain genomic integrity in the face of damage by both endogenous and exogenous alkylating agents. However, paradoxically, bacterial and mammalian AGTs have been shown to increase cytotoxicity and mutagenicity of dihaloalkanes and other bis-electrophiles when expressed in bacterial cells. We have extended these studies to mammalian cells using CHO cells that lack AGT expression and CHO cells stably transfected with a plasmid that expresses human AGT. The cytotoxicity of 1,2-dibromoethane, dibromomethane and epibromohydrin was significantly increased by the presence of AGT but cytotoxicity of butadiene diepoxide was not affected. Mutations caused by these agents were assessed using hypoxanthine-guanine phosphoribosyltransferase (HPRT) as a reporter gene. There was a small (c. 2-3-fold) but statistically significant AGT-mediated increase in mutations caused by 1,2-dibromoethane, dibromomethane and epibromohydrin. Analysis of the mutation spectrum induced by 1,2-dibromoethane showed that the presence of AGT also altered the types of mutations with an increase in total base substitution mutants due to a rise in transversions at both G:C and A:T sites. AGT expression also led to mutations arising from the transcribed strand, which were not seen in cells lacking AGT. Although the frequency of deletion mutations was decreased by AGT expression, the formation of large deletions (> or = 3 exons) was increased. This work demonstrates that interaction of AGT with some bis-electrophiles can cause mutagenicity and diminished cell survival in mammalian cells. It is consistent with the hypothesis that DNA-AGT cross-links, which have been characterized in experiments with purified AGT protein and such bis-electrophiles, can be formed in mammalian cells.

Alkyltransferase-mediated toxicity of 1,3-butadiene diepoxide.

Human O(6)-alkylguanine-DNA alkyltransferase (hAGT) expression increases mutations and cytotoxicity following exposure to 1,3-butadiene diepoxide (BDO), and hAGT-DNA cross-links are formed in the presence of BDO. We have used hAGT mutants to investigate the mechanism of cross-link formation and genotoxicity. Formation of a hAGT-DNA conjugate in vitro was observed with C145S and C145A mutant proteins but was considerably diminished with the C145A/C150S double mutant confirming that cross-linking primarily involves either of these two cysteine residues, which are located in the active site pocket of the protein. Cross-link formation by BDO occurred both via (a) an initial reaction of BDO with hAGT followed by attack of the reactive hAGT complex on DNA, and (b) the initial reaction of BDO with DNA followed by a reaction between hAGT and the DNA adduct. These results differ from those with 1,2-dibromoethane (DBE) where Cys(145) is the only site of attachment and pathway (b) does not occur. The complex formed between hAGT at Cys(145) and BDO was very unstable in aqueous solution. However, the BDO-hAGT complex at Cys(150) exhibited stability for more than 1 h. The effect of hAGT and mutants on BDO-induced genotoxicity was studied in E. coli using the forward assay to rifampicin resistance. Both mutations and cell killing were greatly increased by wild type hAGT, and there was a smaller but significant effect with the C145A mutant. The R128A mutant and R128A/C145A and C145A/C150S double mutants were ineffective, supporting the hypothesis that the formation of hAGT-DNA cross-links is responsible for the enhanced genotoxicity detected in this biological system. In the absence of hAGT, there were equal proportions of G:C to A:T transitions, G:C to T:A transversions, and A:T to T:A transversions. Wild type hAGT expression yielded significantly greater G:C to A:T and A:T to G:C transitions, whereas C145A mutant expression resulted in more transitions and transversions at A:T base-pairs.

Sexually acquired acute hepatitis C infection diagnosed during pregnancy: a case report of successful postpartum treatment.

BACKGROUND: Infection with the hepatitis C virus (HCV) during pregnancy has emerged as an increasingly recognized and prevalent condition among women of reproductive age in the United States. While screening recommendations exist for pregnant women at high risk of HCV infection, pregnant women with HCV are often under-screened, not diagnosed, or do not receive adequate follow-up, thereby increasing the risk of suboptimal maternal and infant outcomes (including in future pregnancies). CASE: A pregnant woman living with HIV presented with intrahepatic cholestasis of pregnancy. She had tested negative for HCV earlier in pregnancy as part of routine screening recommended for women living with HIV. She was found to have sexually acquired a new HCV infection from her partner during pregnancy. She successfully completed treatment postpartum. CONCLUSION: With the rise in HCV infection among pregnant patients, physicians should be diligent in assessing pregnant women and their partners for HCV risk factors, testing for HCV when risk is identified, and arranging follow-up testing and treatment for HCV-positive mothers and their infants.

Incidence of Acute Kidney Injury in Patients Coinfected with HIV and Hepatitis C Virus Receiving Tenofovir Disoproxil Fumarate and Ledipasvir/Sofosbuvir in a Real-World, Urban, Ryan White Clinic.

Ledipasvir/sofosbuvir (LDV/SOF), an antiviral treatment for hepatitis C virus (HCV), and tenofovir disoproxil fumarate (TDF), an antiretroviral for treating human immunodeficiency virus (HIV), may be coadministered in patients coinfected with these viruses. A drug interaction between LDV and TDF could increase TDF-associated nephrotoxicity rates; however, there is minimal clinical evidence describing acute kidney injury (AKI) rates in this population. This study was conducted at a Ryan White-funded facility in Atlanta, Georgia, that cares for over 5,000 patients with AIDS. This retrospective cohort used chart review to assess occurrence of and risk factors for AKI in HIV/HCV-coinfected patients receiving LDV/SOF and antiretroviral therapy (ART). AKI rates were compared between TDF-containing and non-TDF-containing ART groups according to Kidney Disease Improving Global Outcomes (KDIGO) criteria. Additional evaluated risk factors for AKI included chronic kidney disease and use of boosted protease inhibitor-based ART. In the 117 included patients, the overall incidence of AKI was 27.3%. AKI occurred more frequently in the non-TDF group (13/86, 15.1% vs. 19/31, 61.3%, p < .001). All AKI was KDIGO stage 1. From multivariable logistic regression, the only independent predictor of AKI was treatment with non-TDF relative to TDF (adjusted odds ratio 6.51, 95% confidence interval 2.34-18.10, p < .001). In this real-world cohort of HIV/HCV-coinfected patients, KDIGO-defined AKI was common, but occurred less frequently in patients receiving TDF-based ART. Our study suggests that patients with normal baseline renal function can be safely treated with TDF and LDV/SOF without significant nephrotoxicity if renal function is closely monitored.

Chronic hepatitis C virus infection and subsequent HIV viral load among women with HIV initiating antiretroviral therapy.

OBJECTIVES: One in four persons living with HIV is coinfected with hepatitis C virus (HCV). Biological and behavioral mechanisms may increase HIV viral load among coinfected persons. Therefore, we estimated the longitudinal effect of chronic HCV on HIV suppression after ART initiation among women with HIV (WWH). DESIGN: HIV RNA was measured every 6 months among 441 WWH in the Women's Interagency HIV Study who initiated ART from 2000 to 2015. METHODS: Log-binomial regression models were used to compare the proportion of study visits with detectable HIV RNA between women with and without chronic HCV. Robust sandwich variance estimators accounted for within-person correlation induced by repeated HIV RNA measurements during follow-up. We controlled for confounding and selection bias (because of loss to follow-up and death) using inverse probability-of-exposure-and-censoring weights. RESULTS: One hundred and fourteen women (25%) had chronic HCV before ART initiation. Overall, the proportion of visits with detectable HIV RNA was similar among women with and without chronic HCV [relative risk (RR) 1.19 (95% CI 0.72, 1.95)]. Six months after ART initiation, the proportion of visits with detectable HIV RNA among women with chronic HCV was 1.88 (95% CI 1.41-2.51) times that among women without HCV, at 2 years, the ratio was 1.60 (95% CI 1.17-2.19), and by 6 years there was no difference (1.03; 95% CI 0.60-1.79). CONCLUSION: Chronic HCV may negatively impact early HIV viral response to ART. These findings reaffirm the need to test persons with HIV for HCV infection, and increase engagement in HIV care and access to HCV treatment among persons with HIV/HCV coinfection.

Highly Successful Hepatitis C Virus (HCV) Treatment Outcomes in Human Immunodeficiency Virus/HCV-Coinfected Patients at a Large, Urban, Ryan White Clinic.

BACKGROUND: The introduction of direct-acting antivirals (DAAs) created a major paradigm shift in the treatment of chronic hepatitis C. Currently, there is little "real-world" data regarding hepatitis C virus (HCV) treatment outcomes in the human immunodeficiency virus (HIV)/HCV-coinfected population. METHODS: This retrospective cohort study examined HCV treatment outcomes of HIV/HCV-coinfected patients at a large, urban, Ryan White-funded clinic caring for an underserved population. All HIV patients initiating HCV treatment from January 1, 2013 to November 30, 2015 were included in the analysis. The primary end point was sustained virologic response 12 weeks after the end of therapy (SVR12). RESULTS: A total of 172 patients initiated HCV treatment within the study period: 79% were male, 83% were black, 95% were HCV genotype 1, 79% were HCV treatment naive, and 16% had cirrhosis. At baseline, median CD4 was 494 cells/µL (interquartile range, 316-722) and 92% had HIV ribonucleic acid less than 40 copies/mL. The most common DAA initiated was ledipasvir/sofosbuvir (LDV/SOF) (85%), with 92% receiving 12 weeks of treatment. Overall, SVR12 was 93% by intention-to-treat analysis and 98% by per-protocol analysis. The majority of patients on LDV/SOF did not report any adverse effect. One patient in the ribavirin plus SOF group discontinued treatment due to adverse effect. CONCLUSIONS: In a cohort of mainly black, male, HIV/HCV-coinfected patients at a large, urban, Ryan White clinic, HCV treatment with DAAs resulted in high SVR12 rates and was well tolerated despite real-world challenges including medication access barriers and drug interaction concerns.

Experience with Kaposi Sarcoma Herpesvirus Inflammatory Cytokine Syndrome in a Large Urban HIV Clinic in the United States: Case Series and Literature Review.

In 2010, a new entity, characterized by the classical signs and symptoms of Kaposi sarcoma herpesvirus-associated multicentric Castleman's disease (KSHV-MCD) in the absence of pathologic evidence of MCD, was described in individuals living with HIV. This syndrome was named KSHV inflammatory cytokine syndrome (KICS). It carries mortality rates of up to 60%. To date, there are no standard therapies. Treatment regimens studied in clinical trials for MCD disease are used in cases of KICS.

Antiretroviral Therapy for Prevention of Human Immunodeficiency Virus Infection.

Human immunodeficiency virus (HIV) infection is considered a chronic medical condition. Several new drugs are available, including fixed-dose combination tablets, that have greatly simplified combination antiretroviral therapy (ART) regimens to treat HIV, while increasing the life-expectancy of infected individuals. In the last decade, multiple well-regarded studies have established the benefits of using ART in high-risk, HIV-negative persons to prevent HIV acquisition. The primary care provider must not only understand commonly encountered issues pertaining to ART, such as toxicities and drug interactions, but also needs to be aware of using ART for HIV prevention.

Effect of O6-alkylguanine-DNA alkyltransferase on genotoxicity of epihalohydrins.

The effect of O(6)-alkylguanine-DNA alkyltransferase (AGT) on the toxicity and mutagenicity of epihalohydrins was studied. AGT is a DNA repair protein that protects cells from agents that produce genotoxic O(6)-alkylguanine lesions by transferring the alkyl group to an internal cysteine residue (Cys(145) in human AGT) in a single-step. This cysteine acceptor site is highly reactive and epihalohydrins reacted readily with AGT at this site with a halide order of reactivity of Br > Cl > F. AGT expression in bacterial cells caused a very large increase in the mutagenicity and cytotoxicity of epibromohydrin. The mutations were almost all G:C to A:T transitions. Epichlorohydrin also augmented AGT-mediated mutagenesis but to a lesser extent than epibromohydrin. In vitro experiments showed that AGT was covalently cross-linked to DNA in the presence of epibromohydrin and that this conjugation occurred predominantly at Cys(145), and to a smaller extent at Cys(150), a less reactive residue also located within the active site pocket. Two pathways yielding the AGT-DNA adduct were found to occur. The predominant mechanism results in an AGT-epihalohydrin intermediate, which, facilitated by the DNA binding properties of AGT, then reacts covalently with DNA. The second pathway involves an initial reactive DNA-epihalohydrin intermediate that subsequently reacts with AGT. Our results show that the paradoxical AGT-mediated increase in genotoxicity which has previously been shown to occur with dihaloalkanes, butadiene diepoxide and nitrogen mustards, also occurs with epihalohydrins and is likely to contribute to their toxicity and mutagenicity.