Alcohol consumption enhances antiretroviral painful peripheral neuropathy by mitochondrial mechanisms

Abstract

A major dose-limiting side effect of human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) chemotherapies, such as the nucleoside reverse transcriptase inhibitors (NRTIs), is a small-fiber painful peripheral neuropathy, mediated by its mitochondrial toxicity. Co-morbid conditions may also contribute to this dose-limiting effect of HIV/AIDS treatment. Alcohol abuse, which alone also produces painful neuropathy, is one of the most important co-morbid risk factors for peripheral neuropathy in patients with HIV/AIDS. Despite the prevalence of this problem and its serious impact on the quality of life and continued therapy in HIV/AIDS patients, the mechanisms by which alcohol abuse exacerbates highly active antiretroviral therapy (HAART)-induced neuropathic pain has not been demonstrated. In this study, performed in rats, we investigated the cellular mechanism by which consumed alcohol impacts antiretroviral-induced neuropathic pain. NRTI 2',3'-dideoxycytidine (ddC; 50 mg/kg) neuropathy was mitochondrial-dependent and PKCε-independent, and alcohol-induced painful neuropathy was PKCε-dependent and mitochondrial-independent. At low doses, ddC (5 mg/kg) and alcohol (6.5% ethanol diet for 1 week), which alone do not affect nociception, together produce profound mechanical hyperalgesia. This hyperalgesia is mitochondrial-dependent but PKCε-independent. These experiments, which provide the first model for studying the impact of co-morbidity in painful neuropathy, support the clinical impression that alcohol consumption enhances HIV/AIDS therapy neuropathy, and provide evidence for a role of mitochondrial mechanisms underlying this interaction.

Figure 1. Mechanical hyperalgesia induced by the combination of ddC and ethanol diet (ED)

Animals were fed ethanol (■; Δ) or control (▼; ◇) diet for four days. Low dose of ddC (5 mg/kg; i.v.; ■; ▼) or vehicle (Δ; ◇) was injected on the fourth day. Rats treated with ddC (▼) or ED (Δ) alone did not show significant changes in mechanical nociceptive threshold. However, ED rats that received a single injection of ddC (■) showed rapid onset mechanical hyperalgesia that was still present, without attenuation, on the 8^th day. The three-way repeated measures ANOVA showed a significant time × diet × drug group interaction (F_4,80=2.509, p=0.048). The follow-up 2-way repeated measures ANOVA comparing ddC with vehicle in animals with ED showed a significant drug × time interaction (F_4,40=4.496; p<0.001), as well as a significant main effect of drug (F_1,10=14.016; p=0.004). The follow-up 2-way repeated measures ANOVA comparing ddC with vehicle in animals with the control diet showed no significant interactions of main effects. N=6 paws for all groups.

Figure 2. Antagonism of hyperalgesia induced by ddC, ethanol diet (ED) or the combination of ddC and ED

The effect of the non-specific caspase inhibitor Z-VAD-FMK (ZVAD, 5μg/5μl), the mitochondrial electron transport complex (mETC) selective inhibitors rotenone (complex I, 5μg/5μl) and oligomycin (complex V, 5μg/5μl), the antioxidant α-lipoic acid (5μg/5μl), the ATP-dependent mechanisms antagonist (Ap4A, 5μg/5μl) or the PKCε-specific translocation inhibitor peptide (PKCε-I, 1μg/5μl) on mechanical hyperalgesia was tested in the three experimental models. All inhibitors were injected intradermally into the hind paw at the site of nociceptive testing and the mechanical withdrawal threshold evaluated 30 minutes after their injection. (A) Rats were treated with a single intravenous injection of ddC (50 mg/kg). The inhibitors were tested five days later. The one-way ANOVA was significant (F_6,35=20.122; p<0.001). Scheffé post-hoc test showed that all groups except PKCε-I were significantly different from the vehicle control group (all *p<0.001); (B) Rats were fed ED during three weeks in a regimen of 4 days with ED/3 days normal diet. The inhibitors were tested at the end of the third week. The one-way ANOVA was significant (F_6,35=11.024; p<0.001). Scheffé post-hocs showed that only the $\mathrm{C}\widetilde{\epsilon -\mathrm{I}}$ group was significantly different from the vehicle control group (*p<0.001); (C) Rats were fed ED for four days and, on the fourth day a low dose of ddC (5 mg/kg; i.v.) was administered. The inhibitors were tested 24 hours later. The one-way ANOVA was significant (F_6,35=30.772; p<0.001). Scheffé post-hocs showed that the vehicle control was significantly different from all groups (*p<0.001) except the ZVAD and the $\mathrm{C}\widetilde{\epsilon -\mathrm{I}}$ groups (p=0.709 and p=0.612, respectively). Paw withdrawal threshold was evaluated by the Randall-Selitto paw withdrawal test. All groups N=6.

Figure 3. PKCε independence of hyperalgesia induced by the combination of ddC and ED

Treatment with ODN antisense for PKCε mRNA (AS) or mismatch (MM), started 3 days before ethanol diet (ED) and continued until the last day of ED (4^th day). ddC was intravenously injected into the tail on the last day of ED; the hind paw mechanical withdrawal threshold was evaluated 24 hours later. Control experiment (two right bars) was performed in rats submitted to ED for 2 weeks (4 days with ED/3 days normal diet) and treated with AS for PKCε mRNA or MM for 3 days before the evaluation for the presence of hyperalgesia. Hind paw mechanical withdrawal threshold was evaluated by the Randall Selitto paw withdrawal test. Two-way ANOVA demonstrated a significant interaction (F_1,20=12.431; p=0.002). In order to determine the basis of this interaction the responses to the AS and MM treatments were compared separately for the ED+ddC group and for the control (ED, 2 weeks) group. For the control group, the AS treatment differed significantly from the MM treatment (F_1,10=34.967; *p<0.001), but for the ED+ddC group, the AS and MM treatments did not differ significantly (F_1,10=1.687; p=0.223). N=6 paws for all groups.

Figure 4. Interruption of ethanol diet (ED) does not reverse low-dose-ddC-induced mechanical hyperalgesia

Animals were submitted to ED for one (panel A) or two (panel B) weeks, in a regimen of 4 days with ED/3 days normal diet. Single low dose of ddC (5 mg/kg; ■) or vehicle (o) was injected intravenously into the tail four days after ED was begun. Twenty-four hours later, the ED+ddC group showed decreased hind paw mechanical threshold. ED was interrupted in different time points (after one or two weeks) and, the mechanical hyperalgesia, evaluated 1, 3, 4 ,5, 8, 9, 12, 15, 16 and 24 days after the first day of ED. Two repeated measures ANOVAs demonstrated that the groups that received ddC (■) were significantly different from the groups that received vehicle (o) in both panels: time × treatment interaction was (Panel A, F_9,90=8.906; p< 0.001; Panel B, F_9,90=5.304; p<0.001), main effect of group was (Panel A, F_1,10=18.810; p= 0.001; Panel B, F_1,10=19.054; p=0.001). N=6 paws for all groups.