The present study evaluated liver mitochondria in AZT-treated B6C3F1
mice to understand the role of mitochondria in sex-related differences in the development of lactic acidosis. Both male and female B6C3F1
mice were treated with 400, 500, or 600
mg/kg/d AZT at doses predicted to severely alter mitochondrial function leading to hyperlactataemia/lactic acidosis. Despite the relatively high doses of AZT, there was no increase in plasma lactate levels in these mice. However, other classical parameters of clinical toxicity observed with AZT were altered and in a sex-related manner.
During the course of AZT treatments, hematological parameters were measured through 11 weeks of treatment to monitor possible anemia in these mice. Following 11-week exposure, significant changes in hematological parameters were found at all AZT exposures
(). An AZT-related decline in the RBC count was observed in both sexes. The decreases in the RBC counts were 13%, 11%, and 8% in male AZT-treated mice at 400, 500, and 600
mg/kg/d, respectively, whereas the decreases were between 16–26% in female mice compared to 0-week (), and these declines were significant at 400 and 600
mg/kg/d AZT only in female mice. This may indicate a greater influence of AZT on the RBC count in female mice than males and this sex-related difference was significant at the 500
mg/kg/d dose. The occurrence of anemia characterized by reduced Hb levels has been reported in HIV-1 infected patients on NRTI therapies [24
]. In the present study, however, the Hb contents were not significantly altered despite the decline in RBC counts at all AZT doses (). On the other hand, small increases in RBC and Hb contents were observed in male and female mice following 11-week vehicle treatment compared to 0-week, and the increase was significant only in RBC counts in male mice (). At present, a likely mechanism underlying significant findings in vehicle-treated mice is unclear. More experiments need to be conducted to evaluate these changes.
Levels of hematological parameters in B6C3F1 mice.
In addition to the decline in RBC counts, AZT-treated mice showed significant dose-related increases in MCV and MCH levels
(). Male mice treated with 400, 500, or 600
mg/kg/d AZT had a 22%, 22%, or 29% increase, respectively, in MCV at 11-week compared to 0-week. In AZT-treated female mice, the MCV levels were increased by 29%, 29%, and 36% following 11-week treatment with 400, 500, and 600
mg/kg/d AZT, respectively. In both AZT-treated male and female mice, these changes in MCV were associated with increases in MCH levels. While male mice had 15%, 17%, and 22% increases in MCH at 400, 500, and 600
mg/kg/d AZT, respectively, female mice showed 19%, 20%, and 27% increases at 400, 500, and 600
mg/kg/d AZT, respectively. Thus, as for the RBC counts, a greater AZT influence was seen in female mice, and this sex-related difference was significant at 400 and 500
mg/kg/d doses for both MCV and MCH levels but not at 600
mg/kg/d (). It is possible that beyond a certain threshold high concentration of AZT may overcome the genomic differences between sexes.
The MCV represents the average size of the RBC, and MCH indicates the amount of Hb in each RBC. Dose-related increases in MCV and MCH levels in AZT-treated mice might suggest a compensatory mechanism in response to a dose-related decline in the RBC count, which may further explain nonsignificant changes observed in Hb contents in these mice. Corroborating these findings are animal studies that reported hematologic toxicity as evidenced by the decreased erythrocyte count, higher MCV and MCH levels, and increased incidence of macrocytic anemia during AZT exposure [26
]. The increased MCV indicates larger than normal RBCs, which are known as macrocytes. Although Hb levels were not altered in AZT-treated mice, higher MCV and MCH levels in response to decreased RBC count may indicate the initial phase of macrocytic anemia in these mice. Interestingly, a sex effect was seen in both MCV and MCH levels, where AZT-treated female mice had greater increases than their male counterparts and these increases were significant at 400 and 500
mg/kg/d doses. Altogether, these findings suggest the occurrence of hematotoxicity in AZT-treated B6C3F1
mice at all doses and this toxicity appeared to be greater in female mice than in males.
Although an increase in plasma lactate levels was anticipated upon AZT exposure, 12-week dosing with AZT significantly reduced plasma lactate levels in both male and female mice compared to the levels before treatment began
(). The decreases in lactate levels showed no AZT dose response and were also found in animals treated with vehicle alone. The mechanism(s) underlying these decreases in lactate levels is unclear. Control and treated mice were given McTween, which is a commonly used emulsifying agent in drug treatments of laboratory animals in toxicology studies. However, changes in lactate levels in vehicle-treated mice similar to those seen in AZT-treated mice suggest a likely influence of McTween on lactate levels rather than the AZT. In future studies, the use of sterile water as the drug vehicle may help avoid this possible confounding effect.
Plasma lactate levels in B6C3F1 mice.
In toxicological studies, increased lactate levels have been related to mitochondrial dysfunction in various drug-induced toxicities [29
]. In the present study, however, none of the AZT doses significantly altered the transcription levels of genes involved in mitochondrial energy production in the liver of B6C3F1
mice. In view of these findings, lack of increase in plasma lactate levels in AZT-treated mice is not surprising. A moderate, but significant effect of AZT was evident only at 600
mg/kg/d on the expression levels of genes associated with apoptosis and lipid metabolism
(). Expression levels of proapoptotic genes
, and Bnip3
) were significantly increased in the liver. Bbc3 induces a conformational change in Bax, which is essential for apoptotic events mediated through the intrinsic pathway [31
], whereas Bnip3 induces apoptosis by opening the mitochondrial permeability pore with consequential membrane depolarization [32
]. Although immunohistochemical evaluation was not performed in the liver tissues to verify apoptotic changes, increased expression of proapoptotic genes may indicate a cellular response to AZT exposure, probably to remove damaged cells. Supporting this interpretation are in vitro
and in vivo
studies that reported increased apoptosis during exposures to antiretroviral drugs, including AZT [33
Table 3 Effect of AZT (600mg/kg/d) on the expression levels of genes in the liver of B6C3F1 mice.
AZT-treated mice also exhibited differential expression of genes involved in lipid metabolism at 600
mg/kg/d dose. Increased transcriptional level of acyl-coenzyme A synthase
) was associated with decreased expression levels of acyl-coenzyme A dehydrogenases for short-chain
), short/branched chain
), and long-chain fatty acids
). Acsl3 is required for the activation of long-chain fatty acids, whereas Acads, Acadsb, and Acadl are involved in the initial step of fatty acid β
-oxidation. Decreases in the expression levels of acyl-coenzyme A dehydrogenases in AZT-treated mice were modest; however, in long-term therapies, such changes may, in part, contribute to fat accumulation in the liver. Hepatic steatosis, a condition characterized by excess fat accumulation within liver cells, has been indicated as one of the complications of antiretroviral therapy in HIV-1 infected patients [35
]. Overall, the effect of AZT on hepatic mitochondria was minimal in B6C3F1
mice. This was further supported by nonsignificant changes in mtDNA copy number in the livers of AZT-treated mice
(). Expression levels of mtDNA-encoded genes critical in energy production also remained unaltered by AZT treatment
(). In addition, AZT-treated mice did not show sex-related differences in the expression levels of mitochondria-related genes or mtDNA copy number in the liver. Sex-based differences in the expression levels were evident only in vehicle-treated controls: female mice showing higher expression levels compared to males, and these genes were associated with complexes I, III, and V of oxidative phosphorylation and the Krebs cycle (data not shown).
Relative mitochondrial DNA (mtDNA) copy number in the livers of B6C3F1 mice.
In contrast to the paucity of gene expression changes found in this investigation, a previous study from our laboratory using a different mouse model showed that the expression of 164 genes was significantly altered by AZT exposure [13
]. The reasons for this dramatic difference may be attributed to differences in genotype, age, and exposure to AZT. In the previous study, a p53 haplodeficient mouse strain was used. The single functional copy of the p53 gene in this strain may allow cells with damaged mitochondria to survive, and thus express altered mitochondria-encoding genes, by partially inhibiting apoptosis. The ages of the mice in the two studies, as well as the duration of exposure, were also different with young animals being used in the prior study (exposed perinatally from gestation day 12 to 18 followed by continuation of same treatment from postnatal day 1 through 28 days of age) and adult animals
(exposed from 8 weeks of age to 20 weeks of age) used in the present study. Further studies will be required to fully understand the possible influence of genotype and age on gene expression changes induced by AZT.
In conclusion, the AZT doses used in the present study, although as high as 600
mg/kg/d, did not induce hyperlactataemia/lactic acidosis in B6C3F1
mice. This could be due to the absence of an AZT effect on hepatic mitochondria as evidenced by nonsignificant changes in the expression levels of mitochondria-related genes: in particular, genes associated with oxidative phosphorylation or the copy number of mtDNA. It is possible that higher doses of AZT and/or longer duration of AZT exposure may be necessary to significantly impair hepatic mitochondrial function in order to develop hyperlactataemia/lactic acidosis in B6C3F1
mice. Alternatively, use of a more sensitive mouse model, such as the p53 haplodeficient mouse, may accelerate mitochondrial dysfunction and will provide better insights into the role of mitochondria in sex-associated differences in AZT-related adverse events. In B6C3F1
mice, AZT effect on mitochondria was limited to modest changes in the expression levels of genes associated with apoptosis and lipid metabolism and occurred only at the highest dose. A striking finding, however, was a sex-related difference in hematologic toxicity in AZT-treated mice: female mice exhibiting a greater response than males.