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Depletion of Mitochondrial DNA in Liver Under Antiretroviral Therapy With Didanosine, Stavudine, Ulrich A. Walker,1 Jochen B¨auerle,1 Montse Laguno,2 Javier Murillas,2 Stefan Mauss,3 G ¨unther Schmutz,3 Bernhard Setzer,1 Rosa Miquel,2 Jos´e M. Gatell,2 and Josep Mallolas2 The “D drug” HIV reverse-transcriptase inhibitors zalcitabine, didanosine, and stavudine
are relatively strong inhibitors of polymerase-gamma compared with the “non–D drugs”
zidovudine, lamivudine, and abacavir. D drugs deplete mitochondrial DNA (mtDNA) in
cultured hepatocytes. This mtDNA depletion is associated with an increased in vitro pro-
duction of lactate. To investigate the origin of hyperlactatemia in HIV-infected patients and
the effects of antiretroviral therapy on liver mtDNA, we biopsied liver tissue from 94
individuals with chronic hepatitis C virus (HCV) infection. Eighty subjects were coinfected
with HIV. Serum lactate was measured at the time of biopsy. Hepatic mtDNA and liver
histology were centrally assessed. Liver mtDNA content of HIV-infected patients receiving D
drugs at the time of biopsy (n ؍ 34) was decreased by 47% (P<.0001) compared with those
without D drugs (n ؍ 35). Aside from a possible association between HCV genotype I status
and mtDNA depletion in multivariate analysis, there were no other virologic, immunologic,
histologic, demographic or treatment-related variables that could explain the mtDNA de-
pletion. Lactate was above the upper limit of normal in only three patients, all of whom were
treated with D drugs. The mtDNA in each of them was lower than in any non–D drug patient
and significantly (P ؍ .017) depleted compared with D drug patients with normal lactate. In
conclusion, D drug treatment is associated with decreased hepatic mtDNA in HIV-infected
patients with chronic HCV infection. Moderate mtDNA depletion in liver does not neces-
sarily lead to hyperlactatemia, but more pronounced decreases in hepatic mtDNA may be an
important contributor to lactate elevation. (HEPATOLOGY 2004;39:311–317.)
Antiretroviraltherapy(ART)hassignificantlyde- tease inhibitors (PIs) or a nonnucleoside reverse creased the HIV-associated morbidity and mor- transcriptase inhibitor (NNRTI), or of three NRTIs.2 tality in industrialized countries.1 ART usually With prolonged exposure to antiretroviral drugs, clini- consists of a combination of two nucleoside analogue re- cians became aware of long-term side effects of individual verse transcriptase inhibitors (NRTIs) with either pro- ART components. Many adverse effects of the NRTIclass of anti-HIV drugs are now related to the fact thatNRTIs undergo intracellular triphosphorylation, then in- Abbreviations: ART, antiretroviral therapy; NRTI, nucleoside analogue reverse transcriptase inhibitor; PI, protease inhibitor; NNRTI, nonnucleoside reverse tran- hibit the replication of mitochondrial DNA (mtDNA) by scriptase inhibitor; mtDNA, mitochondrial DNA; ddC, zalcitabine; ddI, di- interacting with gamma-polymerase.3 In vitro studies danosine; d4T, stavudine; HCV, hepatitis C virus; nDNA, nuclear DNA; ULN, point toward differences between the potencies of the upper limit of normal; ALT, alanine aminotranferase. From 1Medizinische Universita¨tsklinik, Department of Clinical Immunology, individual NRTIs in depleting mtDNA, with the so- Freiburg, Germany; 2Hospital Clinı´c, Barcelona, Spain; and the 3Center for HIV called “D drugs” zalcitabine (ddC), didanosine (ddI), and and Hepatogastroenterology, Du¨sseldorf, Germany. stavudine (d4T) being relatively strong inhibitors of poly- Received August 22, 2003; accepted November 24, 2003.
This work was supported by the BMBF, Kompetenznetz HIV/AIDS (grant num- merase-gamma compared with the other currently li- censed nucleoside analogues (so-called “non–D drugs”).4,5 Address reprint requests to: Ulrich A. Walker, M.D., Medizinische Universi- Studies performed in vitro and in animals suggest that ta¨tsklinik, Department of Clinical Immunology, Hugstetterstr. 55, D-79106Freiburg, Germany. E-mail: [email protected]; fax: depletion of mtDNA may represent an underlying mech- anism of NRTI-related hepatic side effects in HIV pa- Copyright 2004 by the American Association for the Study of Liver Diseases. tients.5–7 Cell models and animal data, however, have Published online in Wiley InterScience (www.interscience.wiley.com).
DOI 10.1002/hep.20074 limitations in predicting clinical toxicities, partly because of pharmacokinetic differences between species and partly controlled diabetes mellitus, end-stage renal disease, or because of variations in the uptake and phosphorylation severe respiratory disease. Group 1 was subdivided into of nucleosides into tissues, cells, and mitochondria. To three subgroups based on the antiretroviral regimen at the date, only limited observational mtDNA data of HIV time of biopsy: patients receiving no antiretroviral ther- patients receiving ART are available,8,9 and a systematic apy (subgroup 1A), no D drug at all (subgroup 1B), or at study has not been conducted. Furthermore, the available data conflict with regard to zidovudine, because mtDNA Group 2 subjects had to meet all the entry criteria of depletion has also been observed despite the fact that this group 1, except for the fact that they were HIV-negative.
NRTI is not a strong inhibitor of gamma-polymerase at They were recruited to assess possible unspecific effects of HIV infection on mtDNA levels in liver.
Slight and asymptomatic elevations of lactate are fre- Liver Histology. Percutaneous liver biopsies were per-
quently associated with the prolonged use of NRTIs and formed using a Mengini (16-gauge) needle. The largest may also be related to mtDNA depletion in tissues.3,10 aliquot of each biopsy cylinder (Ն20 mm) was embedded Such mild hyperlactatemia has to be distinguished from in paraffin and centrally assessed by a single pathologist, lactic acidosis or symptomatic hyperlactatemia, which who was blinded to the clinical and laboratory informa- have been reported in association with liver pathology.11 tion. The degrees of liver fibrosis and necroinflammatory The contribution of liver impairment (particularly of he- activity were scored on Haematoxilin-Eosin and Masson’s patic mtDNA depletion) to both forms of lactate eleva- trichrome stains according to Scheuer’s classification.13 tion is not clear, although results from an exogenous The percentage of hepatocytes displaying signs of macro- lactate challenge test have suggested that asymptomatic or microvesicular steatosis was also scored.
hyperlactatemia may result from a loss of lactate clearance Quantification of mtDNA. The second aliquot of
(which is likely to involve the liver) and increased lactate the liver biopsy (4 mm) was immediately frozen and stored at Ϫ70° C until shipment on dry ice for centralized Our aim was to investigate if NRTIs and especially D and blinded mtDNA measurements by quantitative drugs deplete mtDNA in hepatic tissue of HIV patients Southern blot analysis as described previously.5,14,15 and if there is a link between mtDNA depletion in liver mtDNA was probed with a 12.9-kb pair, random-prime and lactate elevation. Because of ethical difficulties in ob- digoxigenin-labeled fragment, spanning nucleotide posi- taining repeat liver biopsies, we chose a cross-sectional tions 3470 and 16379 of human mtDNA; nuclear DNA design and selected HIV patients with chronic hepatitis C (nDNA) was simultaneously detected with a second virus (HCV) coinfection as the principal study popula- probe, directed against the multicopy 18S ribosomal DNA gene. The intensities of the mtDNA and nDNAsignals were densitometrically quantified using Scion-im- Experimental Procedures
age (Scion Corporation, Frederick, MD), and mtDNAwas normalized for nDNA-content by calculating the Human Subjects. After ethics committee approval,
mtDNA/nDNA ratio. Two DNA standards extracted patients were recruited in the Hospital Clinı´c, Barcelona, from HepG2 hepatoma cells (ATCC HB-8065) and hu- and the HIV Centre, Du¨sseldorf, from February 2001 man fibroblasts were run in parallel on every blot to assess until October 2002. Consecutive patients were enrolled if assay variations. The mtDNA/nDNA measurements they granted informed consent and met the inclusion and were reliable with an interrun variation of 20%; large exclusion criteria for one of the following study groups.
variations in the amount of DNA loaded onto the gel do Group 1 consisted of HIV-1 patients with chronic not influence the result.14,15 Southern blot analysis was HCV coinfection, as confirmed by a positive HCV-anti- also used to screen for large-scale mtDNA deletions.
body ELISA and positive HCV-RNA measurements Other Measurements. At the time of liver biopsy, a
(Ͼ500 IU/mL). Patients in this group had to meet further forearm vein was cannulated. After at least one hour, inclusion criteria such as no interferon and ribavirin pre- blood was drawn from rested patients without the use of a treatment and absence of HbsAg and other liver diseases tourniquet. Patients were instructed to avoid fist clench- (e.g., autoimmune hepatitis, primary biliary cirrhosis, al- ing and hand pumping. Blood was collected in sodium pha-1 antitrypsin deficiency, hepatocellular carcinoma, fluoride/potassium oxalate tubes and placed on ice. Lac- haemochromatosis, and Wilson’s disease). Pregnant or tate was immediately tested enzymatically in an auto- lactating females were excluded, as were patients who con- mated analyzer (Roche/Hitachi 917; Roche, Basel, sumed intravenous or inhaled drugs, drank alcohol exces- Switzerland) according to the manufacturer’s instruc- sively (Ͼ80 g ethanol/d), or suffered from poorly tions. Both laboratories’ normal reference range was 50 to 220 mg/L. Values were calculated as the upper limit of those not given D drugs (14.5 vs. 18.1 years P ϭ .04) and a slightly lower percentage of undetectable HIV-RNA Further evaluation included quantification of serum (71% vs. 94%, P ϭ .03). Both antiretrovirally treated alanine aminotranferase (ALT), HCV, and HIV viral groups did not differ with respect to NNRTI or current loads (COBAS AMPLICOR HCV-MONITOR version PI use; however, patients on D drugs had a longer cumu- 2.0 and COBAS AMPLICOR HIV-MONITOR version lative exposure to total ART and to PIs than their non–D 1.5, Roche Diagnostics, Basel, Switzerland) and of CD4 lymphocyte counts at the time of biopsy. HCV genotype The mean age (43.7 Ϯ 10.8 years) and sex (79% male) of group 2 did not statistically differ from group 1.
Statistics. The laboratory results and demographic
Liver mtDNA. For easier comparison of the quanti-
and clinical parameters among groups were univariately tative mtDNA results, the mean mtDNA/nDNA ratio of compared using ␹2 tests or Fisher exact tests for categori- group 1A was set as 100%. mtDNA levels (Fig. 1) among cal variables and unpaired t-tests, Wilcoxon Mann-Whit- HIV-negative patients with chronic HCV infection ney tests or Kruskal-Wallis ANOVA on the ranks for (group 2: mean mtDNA/nDNA ratio ϭ 114% Ϯ 54%) continuous variables, as appropriate. Those statistical did not differ from group 1A, as did those of HIV-in- analyses were performed using the Sigma Stat for Win- fected patients receiving ART without D drugs (group dows software version 1.0 (Jandel Corporation, San 1B: mean mtDNA/nDNA ratio ϭ 114% Ϯ 46%). How- Rafael, CA). Both univariate and multivariate linear re- ever, when D drugs were used as part of the antiretroviral gression analyses were done using SPSS for Windows soft- treatment, mtDNA levels were reduced by 47% com- ware Release 11.0.0 (SPSS Inc., Chicago, IL). Trends in pared with biopsies from individuals treated without D mtDNA over time on therapy were estimated using an drugs (group 1C: mean mtDNA/nDNA ratio ϭ 60% Ϯ exponential model that allows variation in the rate of 27%, P Ͻ .0001) and reduced by 40% when compared initial decline and subsequent long-term level (asymp- tote). The nonlinear model was fitted by least-squares in The comparison of mtDNA levels among individual SPSS. The flexibility in the rate of decline allows an ap- NRTIs (Table 2) revealed reduced amounts of mtDNA in proximately linear trend should the data suggest this.
livers of patients receiving stavudine, didanosine, or zal-citabine (mtDNA/nDNA ratios of 62%, P ϭ .0004; 44%, P Ͻ .0001 and 51%, P ϭ .04 of group 1A-levels,respectively). In contrast, mtDNA levels among patients Demographics, Virology, and Immunology. Out of
receiving zidovudine, lamivudine, or abacavir were not a total of 94 patients biopsied, 80 subjects had HIV and reduced (mean mtDNA/nDNA ratios of 111%, P ϭ .79; chronic HCV infection (group 1) and 14 patients had 96%, P ϭ .41; 77%, P ϭ .07, respectively). There was no chronic HCV infection but no HIV infection (group 2).
statistical association between the use or nonuse of PIs or Eleven patients had no antiretroviral therapy at the time NNRTIs (at the time of biopsy, or ever) and mtDNA of biopsy (group 1A). Within group 1A, nine patients depletion. Furthermore, there were no significant corre- were naı¨ve to antiretroviral treatment, one female had a lations of mtDNA levels with total or current time on 3-month exposure to zidovudine during pregnancy 18 months prior to biopsy, and one male had interrupted a Six patients were treated with two D drugs at the time PI-containing first line therapy of 42 months duration for of biopsy; all were receiving the combination of stavudine reasons of revised treatment guidelines; his treatment was and didanosine (Fig. 1). The mean mtDNA/n-DNA ratio in these patients was only 41% Ϯ 10%, which represents The remaining 69 HIV- and HCV-coinfected patients 36% of the ratio in D drug–negative patients (P Ͻ had uninterrupted antiretroviral therapy. At the time of .0001). Compared with the 24 patients receiving stavu- biopsy, 35 were treated without D drugs with various dine as the only D drug, the mean mtDNA/nDNA ratio combinations of zidovudine, lamivudine, and abacavir among subjects receiving a combination of stavudine and (group 1B) and 34 were treated with one or two D drugs didanosine was decreased by 53% (P Ͻ .0001).
(i.e., zalcitabine, didanosine, and stavudine) (group 1C).
To further characterize treatment and disease-related There were no statistical differences between the group factors that may influence mtDNA levels in patients re- 1 subgroups with respect to age, sex, HCV genotype, ceiving treatment (group 1B and 1C, n ϭ 69), multivar- HCV viral load, time of known HIV infection, and CD4 iate regression analyses were performed in which mtDNA count (Table 1). The D drug–treated individuals had a levels were considered as the dependent variable. Each of slightly shorter mean time of known HCV infection than the factors presented in Table 1 was considered as a co- Table 1. Demographic, Virologic, and Immunologic Characteristics Among Patients With HIV/HCV Coinfection
P Value
HIV and HCV Coinfection
(no ART; n ؍ 11)
(D drug–negative; n ؍ 35)
(D drug–positive; n ؍ 34)
Demographics, virology, and immunology mtDNA/nDNA ratio (mean of group 1A: 100%) Group variability is calculated as standard deviation.
Abbreviations: NS, not significant; NA, not applicable.
*Two patients had received ART prior to but not at the time of biopsy. Their treatment is discussed in the text.
variate in both univariate and multivariate regressionanalyses. Current use of D drugs was significantly associ-ated with mtDNA levels in a univariate analysis (P Ͻ.001), while no significant associations were detected foruse or duration of current PI therapy (P ϭ .41, P ϭ .47)or NNRTI therapy (P ϭ .12, P ϭ .12). In addition, theeffect of D drug treatment on mtDNA levels was inde-pendent of the duration of current ART (P ϭ .72). Fur-thermore, neither the cumulative time on ART (P ϭ .24)nor the cumulative time on PI (P ϭ .20) or NNRTI (P ϭ.55) had any influence. With regard to demographic andHIV disease-related effects, no significant associationswere detected between mtDNA levels and age (P ϭ .49),gender (P ϭ .73), CD4 T cell count (P ϭ .62), or unde-tectable HIV viral load (P ϭ .60). Similarly, HCV viralload (P ϭ .96), duration of HCV infection (P ϭ .72),evidence of hepatic inflammatory activity (P ϭ .11), andfibrosis (P ϭ .46) were not significantly associated withmtDNA levels. However, there was a trend toward anassociation between HCV genotype 1 and mtDNA levels(P ϭ .09) that was found to be significant after adjustingfor the effect of D drug use in multivariate regressionanalysis (P ϭ .04). Further assessment of D drug use and Fig. 1. mtDNA/nDNA ratio in liver among all subjects (mean of group HCV genotype status in a general linear model analysis 1A: 100%). The horizontal bar represents group means. The patients withlactate above or equal to the ULN are marked with a star.
revealed no evidence for an interaction between these Table 2. Characteristics of Patients With HIV/HCV Coinfection With Regard to the Use of Particular NRTIs
at the Time of Biopsy
Zidovudine
Lamivudine
Abacavir
Stavudine
Didanosine
P
HIV and HCV Coinfection
Group variability is calculated as standard deviation. Patients receiving zalcitabine (n ϭ 2) are discussed in the text.
Abbreviation: NS, not significant.
*The “no NRTI” group was excluded in the statistical comparison (Kruskal-Wallis ANOVA on the ranks).
†Two patients had received ART prior to but not at the time of biopsy. Their treatment is discussed in the text.
variables (P ϭ .71), suggesting that these factors contrib- ULN Ϯ 0.16). Lactate was higher in both treatment ute independently to mtDNA depletion in the liver. No groups (i.e., in patients receiving D drugs and in patients other tested variable contributed significantly to mtDNA without D drugs) compared with subjects without ART depletion in multivariate regression analyses, while the at the time of biopsy (P ϭ .019 and P ϭ .042, respec- association between use of D drugs and mtDNA levels tively). However, there was no statistical difference of remained highly significant after adjusting for all covari- lactate with respect to the D drug status in patients on antiretroviral treatment. The lactate of HIV-negative pa- We found a significant decline in mtDNA over time on current treatment with D drugs (Fig. 2), dropping froman initial mean of 99.4% (SE, 8.0; group 1A mean,100%) to a long-term value estimated as 57.5 % (SE, 5.3;P Ͻ .000004). However, most of this decline occurs inthe first 6 to 7 months, with no evidence of decline be-yond this time (P ϭ .86).
Qualitative mtDNA alterations (e.g., mtDNA dele- Serum Lactate. Only three individuals had a serum
lactate above or equal to the ULN. Among the three sub-groups of HIV/HCV coinfection, lactate was highest un-der D drug treatment (group 1C: 0.60ϫ ULN Ϯ 0.28),followed by D drug–negative subjects (group 1B: 0.50ϫULN Ϯ 0.19) and by patients without antiretroviral Fig. 2. Trends in mtDNA/nDNA ratio over time on D drug therapy treatment at the time of biopsy (group 1A: 0.35ϫ tients with chronic HCV infection (group 2: 0.43ϫ ration of the treatment in groups 1B and 1C. However, if we ULN Ϯ 0.12) did not statistically differ from its HIV had excluded the two patients with prior ART from group 1A, the significance levels of our results would not have Compared with patients without ART, the mean lac- changed, and the association between mtDNA depletion tate was elevated in subjects using didanosine (0.71ϫ and D drug treatment at the time of biopsy in particular ULN, P ϭ .016) and stavudine (0.59ϫ ULN, P ϭ .027), would have still been detectable (P ϭ .0001).
but also in those receiving zidovudine (0.50ϫ ULN, P ϭ We detected a trend (P ϭ .09) toward an association .04), lamivudine (0.52ϫ ULN, P ϭ .02) and abacavir between HCV genotype I for mtDNA depletion that was (0.56ϫ ULN, P ϭ .046). In summary, lactate levels found to be significant after adjusting for the effects of D tended to be elevated among subjects receiving D drugs, drug use. Interestingly, a similar association has been docu- but this was not statistically significant (Table 2).
mented previously with HCV genotype Ib.17 Such HCV- There was no correlation between mtDNA levels and associated mitochondrial injury could be explained by lactate in the group of all 94 HCV-infected individuals or increased oxidative stress or by several other mecha- its subgroups. Stratification of the mtDNA measure- ments into quartiles revealed only a nonsignificant trend No further virologic, immunologic, histologic, demo- toward lactate elevation with mtDNA depletion among graphic, or treatment-related variables contributed signif- patients coinfected with HCV and HIV.
icantly to mtDNA depletion in the multivariate analysis.
The subjects with lactate above or equal to the ULN The conclusions with regard to the association between were all HIV-positive and treated with stavudine at the the use of individual D drugs and mtDNA depletion are time of biopsy; two of the three were given additional limited by the cross-sectional design of the study, in didanosine. The mean mtDNA/nDNA ratio in the liver which the treatment with individual NRTIs is not inde- of these three patients was 38% Ϯ 11% of untreated pendent from each other and is not randomized. This HIV- and HCV-coinfected controls (P ϭ .003). Com- may be illustrated by the fact that most patients treated pared with the remaining patients on D drugs (67% Ϯ with didanosine were also receiving stavudine. However, 28%), mtDNA levels were significantly diminished (P ϭ the higher mtDNA levels in individuals receiving stavu- .017). Furthermore, the mtDNA/nDNA values of pa- dine as the only D drug, compared with those receiving tients with serum lactate above or equal to the ULN stavudine plus didanosine, supports an independent ef- (range: 25%– 47%) were outside the range of mtDNA fect of didanosine. The coadministration of two NRTIs measurements in individuals without antiretroviral ther- was previously observed to have additive or synergistic apy (group 1A range: 49%–150%) or D drugs (group 1B Our model of mtDNA trends over time (Fig. 2) sug- Other Measurements. Between the subgroups of
gests an mtDNA decline during the initial 6 months of D group 1 (groups 1A, 1B, and 1C), there were no differ- drug therapy, with no further decline beyond this time.
ences with regard to the histologic degrees of liver fibrosis, The kinetics of mtDNA loss are initially influenced by the inflammatory activity, macro- or microvesicular steatosis, degree of gamma-polymerase inhibition and presumably or the serum levels of ALT (not shown). There was also no also by the rates of cell division and mtDNA turnover.5 association between macro- or microvesicular steatosis Eventually, stable mtDNA levels arise in accordance with The mean mtDNA/nDNA ratio in the D drug–treated Discussion
HIV patients was 53% lower, compared with patients receiv- This study analyzed the hepatic mtDNA content in ing anti-HIV treatment without D drugs. The question then patients with chronic HIV and HCV coinfection. The arises as to whether or not this relatively moderate mtDNA HIV patients were divided into three subgroups accord- depletion may be functionally relevant, given the fact that ing to their antiretroviral regimen at the time of biopsy.
wild type mtDNA levels in the order of 20% can maintain The major finding is that antiviral therapy with at least almost normal cell function in vitro.19 Several observations one of the D drugs (didanosine, stavudine, and zalcita- indicate that the in vitro threshold of mtDNA levels within bine) is associated with mtDNA depletion in liver, cells may differ from the in vivo situation in a tissue. For whereas no such relation was detected between mtDNA example, anaerobic ATP production by ample glucose sup- levels and treatment with other antiretroviral drugs.
ply in the medium may allow a relatively long cell survival in Two individuals within group 1A were not naı¨ve to anti- vitro despite severe mtDNA depletion. Fibrotic tissue may retroviral treatment. We chose to include these patients in also be more resistant to mtDNA depletion and thus main- our analysis because we also did not select for a specific du- tain some residual mtDNA. Indeed, in situ hybridization studies in patients with proven mitochondrial cytopathies 5. Walker UA, Setzer B, Venhoff N. Increased long-term mitochondrial tox- failed to establish an in vivo threshold necessary for mtDNA icity in combinations of nucleoside analogue reverse-transcriptase inhibi-tors. AIDS 2002;16:2165–2173.
mutations to trigger a biochemical dysfunction in clinically 6. Gaou I, Malliti M, Guimont MC, Letteron P, Demeilliers C, Peytavin I, affected tissue.20 It is also interesting to note that the magni- Degott C, et al. Effect of stavudine on mitochondrial genome and fatty tude of mtDNA depression was similar in adipose tissue of acid oxidation in lean and obese mice. J Pharmacol Exp Ther 2001;297:516 –523.
patients suffering from HIV-associated lipoatrophy14 and in 7. Gerschenson M, Nguyen VT, St Claire MC, Harbaugh SW, Harbaugh hepatic tissue of inherited mtDNA replication defects with JW, Proia LA, Poirier MC. Chronic stavudine exposure induces hepatic mitochondrial toxicity in adult Erythrocebus patas monkeys. J Hum Virol Our investigations do not demonstrate a clear relation- 8. Brivet FG, Nion I, Megarbane B, Slama A, Brivet M, Rustin P, Munnich ship between mtDNA depletion in hepatic tissue and an A. Fatal lactic acidosis and liver steatosis associated with didanosine and increase in serum lactate, but it is important to note that stavudine treatment: a respiratory chain dysfunction? J Hepatol 2000;32: lactate levels were normal in virtually all patients. In contrast 9. Chariot P, Drogou I, de Lacroix-Szmania I, Eliezer-Vanerot MC, Chazaud to previous reports,22 there was no lactate elevation in indi- B, Lombes A, Schaeffer A, et al. Zidovudine-induced mitochondrial dis- viduals receiving stavudine compared with zidovudine, al- order with massive liver steatosis, myopathy, lactic acidosis, and mitochon- though lactate was somewhat higher with D drug treatment drial DNA depletion. J Hepatol 1999;30:156 –160.
in our study. We are unable to determine the exact reasons 10. Gerard Y, Maulin L, Yazdanpanah Y, Tribonniere X, Amiel C, Maurage CA, Robin S, et al. Symptomatic hyperlactataemia: an emerging compli- for this discrepancy, but possible explanations include the cation of antiretroviral therapy. AIDS 2000;14:2723–2730.
HCV status of our principal study population,17 effects on 11. John M, Mallal S. Hyperlactatemia syndromes in people with HIV infec- mitochondria or hepatic tissue unrelated to mtDNA deple- tion. Curr Opin Infect Dis 2002;15:23–29.
12. Leclercq P, Roth H, Bosseray A, Leverve X. Investigating lactate metabo- tion (as demonstrated for zidovudine4,5,23), and additional lism to estimate mitochondrial status. Antivir Ther 2003;6(Suppl. 4):16.
mitochondrial toxicity in extra-hepatic tissues.9,24 Our study 13. Scheuer PJ. Classification of chronic viral hepatitis: a need for reassess- was also not powered to examine associations between hy- perlactatemia and NRTI therapy. The findings therefore do 14. Walker UA, Bickel M, Lu¨tke Volksbeck SI, Ketelsen UP, Schofer H, Setzer B, Venhoff N, et al. Evidence of nucleoside analogue reverse transcriptase not exclude an important contribution of mtDNA levels in inhibitor-associated genetic and structural defects of mitochondria in adi- liver on serum lactate. This is also supported by the observation pose tissue of HIV-infected patients. J Acquir Immune Defic Syndr 2002; that among the D drug–treated subjects, those with a serum 15. Hammond EL, Sayer D, Nolan D, Walker UA, Ronde A, Montaner JS, lactate above or equal to the ULN had a significant mtDNA Cote HC, et al. Assessment of precision and concordance of quantitative depletion compared with those with a normal lactate.
mitochondrial DNA assays: a collaborative international quality assurance We found that antiviral treatment with D drugs— but study. J Clin Virol 2003;27:97–110.
not with non–D drugs—is associated with hepatic mtDNA 16. Thiers V, Jaffredo F, Tuveri R, Chodan N, Brechot C. Development of a simple restriction fragment length polymorphism (RFLP) based assay for depletion in HCV- and HIV-coinfected subjects. We have HCV genotyping and comparative analysis with genotyping and serotyp- evidence that a moderate mtDNA depletion in hepatic tissue ing tests. J Virol Methods 1997;65:9 –17.
alone is not sufficient to cause mild hyperlactataemia, but 17. Barbaro G, Di Lorenzo G, Asti A, Ribersani M, Belloni G, Grisorio B, Filice G, et al. Hepatocellular mitochondrial alterations in patients with that more pronounced mtDNA depletion may represent an chronic hepatitis C: ultrastructural and biochemical findings. Am J Gas- important factor contributing to lactic acidosis.
18. Rust C, Gores GJ. Does hepatitis C cause liver injury by pathways associated with mitochondrial dysfunction? Am J Gastroenterol 1999;94:2003–2005.
BMBF, Kompetenznetz HIV/AIDS (grant number: 19. Attardi G, Yoneda M, Chomyn A. Complementation and segregation behavior of disease-causing mitochondrial DNA mutations in cellularmodel systems. Biochim Biophys Acta 1995;1271:241–248.
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