ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 2008, p. 2825–2830
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 52, No. 8
Impact of Nucleoside Reverse Transcriptase Inhibitors on Mitochondrial
DNA and RNA in Human Skeletal Muscle Cells?
Akihiko Saitoh,1,2* Richard H. Haas,2,3Robert K. Naviaux,2,4Neurita G. Salva,1,2
Justine K. Wong,1,2and Stephen A. Spector1,2
Division of Infectious Diseases,1Department of Pediatrics,2Department of Neuroscience,3and Department of Medicine,4
University of California San Diego, La Jolla, California
Received 1 April 2008/Returned for modification 2 May 2008/Accepted 30 May 2008
We previously reported that 2?,3?-dideoxyinosine (didanosine, or ddI) significantly altered mitochondrial
DNA (mtDNA) in peripheral blood mononuclear cells in human immunodeficiency virus type 1 (HIV-1)-
infected children who had undetectable plasma HIV-1 RNA for more than 2 years while receiving highly active
antiretroviral therapy. This research examines the in vitro effects of nucleoside reverse transcriptase inhibitors
(NRTIs) on mitochondria of human skeletal muscle cells (HSMCs), including myoblasts and differentiated
myotubes. mtDNA, mitochondrial RNA (mtRNA), and mRNA levels for nuclear mitochondrial regulatory
factors were quantified in vitro using HSMCs, including myoblasts and differentiated myotubes, treated with
NRTIs singly and in combination. After 5 days of treatment, mtDNA was significantly decreased in myoblasts
and myotubes treated with ddI (P < 0.001 and P ? 0.01, respectively) and ddI-containing regimens (P < 0.001
and P < 0.001, respectively) compared to levels in untreated cells. mtRNA (MTCYB) was also significantly
decreased in the myoblasts and myotubes treated with ddI (P ? 0.004) and ddI-containing regimens (P <
0.001). Regardless of the NRTI regimens examined, NRTI combinations significantly decreased mtRNA
(MTCO3) in myoblasts and myotubes (P ? 0.02 and P ? 0.01, respectively). No significant differences were
observed for nuclear mitochondrial regulatory factor mRNA in myoblasts or myotubes when treated with
NRTIs (P > 0.07). ddI and ddI-containing regimens significantly decrease mtDNA and mtRNA in HSMCs,
most notably in myoblasts. These findings may be of particular importance in developing countries, where ddI
is widely used for first-line treatment of HIV-infected children.
Nucleoside reverse transcriptase inhibitors (NRTIs) remain
the backbone for the majority of highly active antiretroviral
therapy (HAART) regimens in combination with protease in-
hibitors or nonnucleoside reverse transcriptase inhibitors in
human immunodeficiency virus type 1 (HIV-1)-infected pa-
tients (13). Mitochondrial toxicity, which is believed to result
from depletion of mitochondrial DNA (mtDNA) by NRTIs
interacting selectively with the DNA polymerase ? (POLG)
(21, 25), is a major concern for HIV-infected patients receiving
antiretroviral therapy (20). Mitochondrial dysfunction due to
the depletion of mtDNA is at least partly responsible for var-
ious NRTI-associated adverse effects, including myopathy, car-
diomyopathy, peripheral neuropathy, pancreatitis, hepatic ste-
atosis, lipodystrophy, and in severe cases, lactic acidosis (11).
Several studies have evaluated the impact of NRTIs on
mtDNA using different in vitro models (14). The study of
human skeletal muscle cells (HSMCs) is generally considered
one of the most clinically relevant in vitro indicators of poten-
tial mitochondrial damage in patients, because mitochondrial
toxicity often presents with muscular symptoms, including mus-
cle atrophy, weakness, fatigue, and cardiomyopathy.
Human skeletal muscle myoblasts are precursors of HSMCs
and are committed to become differentiated muscle cells (32).
Once myoblasts have migrated, differentiated into multinucle-
ated skeletal muscle cells, and fused into parallel arrays, they
are referred to as myotubes. A few studies have evaluated the
impact of NRTIs on mtDNA in differentiated myotubes (1, 2);
however, no data are currently available regarding the impact
of NRTIs on mitochondria in myoblasts. The impact of NRTIs
on myoblasts is of particular importance in infants and children
who are actively differentiating myotubes from myoblasts and
carry more myoblasts in their muscles than adults (8, 12).
Beyond the evaluation of mtDNA, levels of mitochondrial
RNA (mtRNA) and mitochondrial regulatory genes can pro-
vide additional information regarding the risk of clinically im-
portant mitochondrial damage. Although decreases in mtRNA
have been found to coincide with the simultaneous upregula-
tion of nuclear genes involved in transcriptional regulation of
mRNA (24), such as the mitochondrial transcriptional factor A
(Tfam) gene, no studies to date have assessed the impact of
NRTIs, singly or in combination, on these indicators of mito-
chondrial function (14).
We previously reported that 2?,3?-dideoxyinosine (didanosine,
or ddI) significantly altered mtDNA in peripheral blood mono-
nuclear cells (PBMCs) of HIV-1-infected children who had un-
detectable plasma HIV-1 RNA for more than 2 years while re-
ceiving HAART (30). This is particularly important for children,
who are potentially more vulnerable than adults to the adverse
effects of antiretrovirals because of their long-term exposure and
the possible negative impact on growth and development (26).
The objective of this study was to investigate the effects of NRTIs,
expression for nuclear mitochondrial regulatory factors in human
skeletal muscle myoblasts and myotubes. We have specifically
* Corresponding author. Mailing address: Division of Infectious
Diseases, Department of Pediatrics, University of California San Di-
ego, 9500 Gilman Drive, La Jolla, CA 92093-0672. Phone: (858) 534-
7258. Fax: (858) 534-7411. E-mail: email@example.com.
?Published ahead of print on 9 June 2008.
particularly deleterious to mitochondria (30).
(Presented in part at the 46th Interscience Conference on
Antimicrobial Agents and Chemotherapy, San Francisco, CA,
September 2006 [poster H-1905].)
MATERIALS AND METHODS
In vitro assays for mitochondrial toxicity using human skeletal muscle myo-
blasts. Proliferating human skeletal muscle myoblasts in a 24-well plate (Lonza,
Walkersville, MD) were used to investigate the impact of NRTIs on mitochon-
dria. The cells were isolated from postgestational quadriceps or psoas muscle.
ddI and 3?-deoxy-2?,3?-didehydrothymidine (stavudine, or d4T) were purchased
from Sigma-Aldrich (St. Louis, MO); and 3?-azido-3?-deoxythymidine (zidovu-
dine, or AZT), 2?,3?-dideoxy 3?-thiacytidine (lamivudine, or 3TC), and abacavir
sulfate (ABV) were purchased from Moravek Biochemicals (Brea, CA). The
concentrations of NRTIs used for this assay were based on the mean peak
steady-state levels in human plasma during antiretroviral therapy (Cmax): ddI,
11.8 ?mol; d4T, 3.6 ?mol; AZT, 7.1 ?mol; 3TC, 8.3 ?mol; and ABV, 3.0 ?mol
(31, 35). The myoblasts were treated with each NRTI and clinically relevant
NRTI combinations, including ddI-d4T, d4T-3TC, AZT-3TC, and AZT-3TC-
ABV. All NRTIs were suspended in sterile water and diluted with the human
skeletal muscle growth medium (Lonza) before being added into the wells to
reach a total volume of 1.0 ml. As a control, myoblasts were incubated with the
medium alone in triplicate. Myoblasts were incubated at 37°C in 5% CO2; the
medium with/without NRTIs was replaced every 2 days. The NRTIs were added
to the cells at 20 to 30% confluence, and cells were harvested on days 2 and 5
before reaching 70 to 80% confluence. Each evaluation of myoblasts was re-
peated five times.
In vitro assays for mitochondrial toxicity using human skeletal muscle myo-
tubes. To derive differentiated myotubes, myoblasts at approximately 70 to 80%
confluence had the media changed to differentiation media containing alpha-mini-
mal essential medium (Gibco, Carlsbad, CA) with 10% fetal calf serum (Gibco) and
penicillin/streptomycin (100 U/ml and 100 ?g/ml, respectively; Gibco) and incubated
for 72 h to achieve complete differentiation. The NRTIs were suspended in sterile
water, diluted with the differentiation media, and added into the wells to a total
with/without NRTIs was replaced every 2 days. The cells were harvested on day 5.
The assays for myotubes were repeated five times.
Evaluation of the cell proliferation. The cell counts in each well were esti-
mated using the cell proliferation reagent WST-1 (Roche Diagnostics, Indian-
apolis, IN). After the cells were treated with NRTIs, 100 ?l of WST-1 was added
to each well and incubated at 37°C in 5% CO2for 1 h. The supernatants were
collected and transferred into 96-well plates in duplicate and quantified by
spectrophotometer at 450 nm. The ratios were calculated based on the absor-
bance values in the cells treated with NRTIs or NRTI combinations compared to
the values in the cells without treatment (control); all assays were performed in
Extraction of genomic DNA and RNA from myoblasts and myotubes. The cells
were washed twice with phosphate-buffered saline (Gibco) and trypsinized using
Trypsin/EDTA solution (Lonza) and trypsin neutralized solution (Lonza) ac-
cording to the manufacturer’s protocol. Trypsinized cells were washed, resus-
pended with phosphate-buffered saline, and divided into two aliquots. Genomic
DNA was extracted using a QIAamp DNA blood mini kit (Qiagen, Valencia,
CA), and the genomic RNA was extracted using a QIAamp RNeasy kit (Qiagen).
Quantitation of mtDNA and nDNA using real-time PCR. The mtDNA and the
nuclear DNA (nDNA) were quantified by real-time PCR using a LightCycler
(Roche Applied Science, Indianapolis, IN) as described previously (4). The
results were expressed as a ratio of the mean mtDNA level to the mean nDNA
level for a given extract in duplicate (mtDNA:nDNA, unit:copies/cell). The mean
mtDNA ratios were calculated based on the mtDNA levels in the cells treated
with NRTIs and NRTI combinations compared to levels in untreated controls.
Quantitation of mtRNA and mRNA for DNA POLG and Tfam using real-time
PCR. We developed novel assays to quantify the expression of mtRNA, evalu-
ating functionally important mtRNA genes: MTCYB (the genes for cytochrome
B of complex III) and MTCO3 (the genes for cytochrome c oxidase subunits III
of complex IV) (24). We also developed novel assays for mRNA for POLG and
Tfam, which are critical factors for determining the levels of mtDNA (7, 17, 21,
25). The mRNA was quantified by a LightCycler (Roche Applied Science) using
specific primers and probes (IT Biochem, Salt Lake City, UT) (Table 1) and
LightCycler RNA master hybridization probes (Roche Applied Science). For
quantitation, standard curves for MTCYB, MTCO3, POLG, and Tfam genes were
generated using serially diluted DNA extracted from PBMCs of HIV-negative
G6PDH (glucose-6-phosphate dehydrogenase) was selected as a housekeeping
gene using a LightCycler h-Housekeeping gene selection set (Roche Applied
Science), because no differences were observed in the G6PDH mRNA levels in
myoblasts or myotubes treated with NRTIs and NRTI combinations compared to
levels in those without treatment (P ? 0.82). For quantitation, a standard curve
for G6PDH was generated using a LightCycler h-G6PDH housekeeping gene set
(Roche Applied Science).
Each assay included standards with predetermined copies of G6PDH and
MTCYB, MTCO3, POLG, and Tfam genes used as a reference. The samples of
different NRTI treatments were assayed at the same time in the same run. Data
TABLE 1. Summary of primers, probes, and PCR conditions for the analysis of mtRNA and mRNA for Tfam and POLG
Summary for mRNA of:
MTCYB MTCO3Tfam POLG
55°C for 600 s
95°C for 30 s
45 cycles of 95°C for 0 s,
55°C for 15 s, 72°C for
55°C for 600 s
95°C for 30 s
45 cycles of 95°C for 0 s,
55°C for 15 s, 72°C for
55°C for 600 s
95°C for 30 s
45 cycles of 95°C for 0 s,
55°C for 15 s, 72°C for
55°C for 600s
95°C for 30s
45 cycles of 95°C for 0 s,
55°C for 15 s, 72°C for
2826SAITOH ET AL.ANTIMICROB. AGENTS CHEMOTHER.
were analyzed by RelQuant software (Roche Diagnostics) and expressed as
ratios of copies of MTCYB, MTCO3, POLG, Tfam and copies of G6PDH. The
mean RNA ratios were calculated as the targeted RNA:G6PDH ratios in the
treated cells and ratios in the cells without treatment. All assays including
different NRTIs and NRTI combinations were run at the same time.
Statistical analyses. Statistical analyses were performed using SPSS 13.0 soft-
ware (Chicago, IL). The Student t test was used for comparison of numerical
variables in two independent groups. The analysis of variance test was used for
the comparison of numerical variables in ?3 independent groups. All P values
calculated were two sided, and a P value of ?0.05 was considered to be statis-
NRTIs did not affect myoblast and myotube cell numbers.
The myoblast and myotube cell counts with or without treat-
ment estimated by the cell proliferation reagent (WST-1) did
not differ between untreated cells and the cells treated with
different NRTIs and NRTI combinations during the 5-day
incubation period (range, 0.84 to 1.08; P ? 1.00).
mtDNA changes between human skeletal muscle myoblasts
and myotubes treated with NRTIs and NRTI combinations.
mtDNA ratios were evaluated in myoblasts treated with NR-
TIs and NRTI combinations (Table 2). On day 2, the mtDNA
ratios in myoblasts treated with ddI (P ? 0.001) and ddI-d4T
(P ? 0.001) were significantly decreased compared to those in
myoblasts without treatment. In contrast, there was an increase
in mtDNA ratios in myoblasts treated with AZT (P ? 0.03)
and AZT-3TC-ABV (P ? 0.02). Other NRTIs and NRTI com-
binations did not change mtDNA ratios significantly (P ? 0.18
On day 5, the declines in mtDNA ratios in myoblasts treated
with ddI and ddI-d4T were significantly lower than those in myo-
blasts without treatment (P ? 0.001 and P ? 0.001, respectively)
(Fig. 1 and Table 2). Similarly, significant increases in mtDNA
ratios were observed in myoblasts treated with AZT-containing
regimens, including AZT-3TC (P ? 0.003) and AZT-3TC-ABV
(P ? 0.02), compared to those in myoblasts without treatment.
Other treatments did not change the mtDNA levels significantly
(P ? 0.30 to 0.66) (Fig. 1 and Table 2).
Next, the impact of NRTIs on mtDNA in differentiated
myotubes was investigated. The mtDNA ratios in myotubes
treated with ddI (0.50 ? 0.19) and ddI-d4T (0.38 ? 0.11) for 5
days were significantly decreased compared to those in un-
treated cells (1.00) (P ? 0.01 and P ? 0.001, respectively). Of
note, the impact of NRTIs was not as significant as the effect in
myoblasts (P ? 0.03 and P ? 0.16, respectively) (Fig. 1). There
were significant differences in mtDNA ratios in myotubes
TABLE 2. mtDNA ratios in myoblasts treated with NRTIs and
Day 2Day 5
0.51 ? 0.15**
1.20 ? 0.27
1.45 ? 0.30*
1.16 ? 0.07
1.19 ? 0.34
0.42 ? 0.10**
1.17 ? 0.27
1.05 ? 0.15
1.25 ? 0.13*
0.22 ? 0.10**
0.98 ? 0.10
1.14 ? 0.19
0.94 ? 0.25
1.07 ? 0.34
0.23 ? 0.12**
1.13 ? 0.26
1.24 ? 0.12**
1.29 ? 0.21*
aFor each experiment, the mean mtDNA ratios were calculated based on the
values in the cells treated with NRTIs and NRTI combinations compared to
values in untreated controls, which were always set as 1.00. *, P ? 0.05; **, P ?
FIG. 1. mtDNA levels in human skeletal muscle myoblasts and myotubes treated with different NRTIs and NRTI combinations. The white bars
indicate the mean values of mtDNA ratios in human skeletal muscle myoblasts treated with NRTIs and NRTI combinations and those without
treatment (No Tx) for 5 days. The black bars indicate the mean values of mtDNA ratios in human skeletal muscle myotubes treated with NRTIs
and NRTI combinations and those without treatment for 5 days. For each experiment, the mean mtDNA ratios were calculated based on the values
in the cells treated with NRTIs and NRTI combinations compared to values for untreated controls, which were always set as 1.00. Each control
or treatment in myoblasts and myotubes was repeated five times. The brackets indicate ? 1 standard deviation.*, P ? 0.05;**, P ? 0.01.
VOL. 52, 2008NRTIs AND MITOCHONDRIAL TOXICITY IN MUSCLE CELLS2827
treated with AZT (P ? 0.03), d4T-3TC (P ? 0.003), and
AZT-3TC (P ? 0.01) but not with the other treatments (P ?
0.21 to 0.56).
mtRNA changes in human skeletal muscle myoblasts
treated with NRTIs. The mtRNA ratios, including those for
MTCYB and MTCO3, in myoblasts treated with NRTIs and
NRTI combinations did not change significantly on day 2 (P ?
0.37 and P ? 0.94, respectively). However on day 5, the
mtRNA ratios for MTCYB in myoblasts treated with ddI
(0.51 ? 0.14; P ? 0.001), ddI-d4T (0.38 ? 0.13; P ? 0.001),
d4T-3TC (0.63 ? 0.17; P ? 0.002), and AZT-3TC (0.74 ? 0.25;
P ? 0.05) were lower than ratios in those without treatment
(1.00) (Fig. 2A). Other treatment combinations did not signif-
icantly change mtRNA expression for MTCYB in myoblasts
(P ? 0.10 to 0.40). Myoblasts treated with ddI-d4T (0.61 ?
0.05; P ? 0.001) and d4T-3TC (0.71 ? 0.13; P ? 0.001) showed
significantly decreased mtRNA ratios for MTCO3 compared to
ratios in untreated cells (1.00) (Fig. 2B). The decline of
mtRNA ratios for MTCO3 was not statistically significant in
myoblasts treated with other drug combinations (P ? 0.12 to
Next, we evaluated the impact of overall NRTI combina-
tions on mtRNA in myoblasts. The mtRNA ratios for MTCYB
in myoblasts treated with NRTI combinations (0.65 ? 0.26)
and those in myoblasts treated with a single NRTI (0.81 ?
0.27) did not differ significantly (P ? 0.08). However, for the
mtRNA ratios for MTCO3, myoblasts treated with NRTI com-
binations both including and excluding ddI were significantly
lower than those in myoblasts treated with a single NRTI
(0.93 ? 0.20) (P ? 0.02). When we compared the mtRNA
ratios for MTCYB and MTCO3 between ddI or d4T alone and
the ddI-d4T combination in myoblasts and myotubes, signifi-
cant additive results were observed in MTCO3 expression be-
tween ddI and ddI-d4T in myoblasts (P ? 0.004) and myotubes
(P ? 0.05) as well as in MTCTB expression between d4T and
ddI-d4T in myoblasts (P ? 0.004) and myotubes (P ? 0.002).
mtRNA changes in human skeletal muscle myotubes treated
with NRTIs. Similar effects of NRTIs and NRTI combinations
on mtRNA were also observed in myotubes; however, the
patterns of decline in mtRNA were different. On day 5, the
mtRNA levels for MTCYB were significantly decreased in myo-
tubes with different treatments (P ? 0.03) (Fig. 2A). The
myotubes treated with ddI (0.66 ? 0.18; P ? 0.004), d4T
(0.80 ? 0.06; P ? 0.001), ddI-d4T (0.45 ? 0.05; P ? 0.001), and
AZT-3TC-ABV (0.74 ? 0.19; P ? 0.02) demonstrated signif-
icant declines in mtRNA ratios for MTCYB compared to ratios
for those without treatment. Similarly, the declines in mtRNA
ratios for MTCO3 were different among myotubes treated with
different treatments (P ? 0.02) (Fig. 2B). Significant declines
were observed in myotubes treated with d4T (0.69 ? 0.11; P ?
0.001), ddI-d4T (0.78 ? 0.12; P ? 0.005), d4T-3TC (0.75 ?
0.12; P ? 0.002), AZT-3TC (0.72 ? 0.16; P ? 0.006), and
AZT-3TC-ABV (0.64 ? 0.17; P ? 0.003). Similarly, the
mtRNA ratios for MTCO3 in myotubes treated with NRTI
combinations were consistently lower than the ratios for myo-
blasts treated with a single NRTI (0.72 ? 0.13 versus 0.91 ?
0.20; P ? 0.01). No differences were observed for the mRNA
ratios for MTCYB between myotubes treated with a single
NRTI and those treated with NRTI combinations (0.77 ? 0.26
versus 0.85 ? 0.16; P ? 0.20).
mRNA expression for POLG and Tfam in myoblasts and
myotubes treated with NRTIs. We evaluated the mRNA ex-
pression for POLG and Tfam in myoblasts and myotubes
treated with NRTIs singly and in combinations. The treatment
did not alter the mRNA ratios for POLG and Tfam on day 2
(P ? 0.62 and P ? 0.62, respectively) and day 5 (P ? 0.79 and
FIG. 2. mtRNA levels in human skeletal muscle myoblasts treated with different NRTIs and NRTI combinations on day 5. (A) MTCYB, the
gene for cytochrome B of complex III. (B) MTCO3, the gene for cytochrome c oxidase subunits III of complex IV. The white bars indicate the
mean values of mtRNA ratios in human skeletal muscle myoblasts treated with NRTIs and NRTI combinations and those without treatment (No
Tx) for 5 days. The black bars indicate the mean values of mtRNA ratios in human skeletal muscle myotubes treated with NRTIs and NRTI
combinations and those without treatment for 5 days. For each experiment, the mean mtRNA ratios were calculated based on the values in the
cells treated with NRTIs and NRTI combinations compared to values for untreated controls, which were always set as 1.00. Each control or
treatment in myoblasts and myotubes was repeated five times. The brackets indicate ? 1 standard deviation.*, P ? 0.05;**, P ? 0.01.
2828SAITOH ET AL. ANTIMICROB. AGENTS CHEMOTHER.
P ? 0.70, respectively) in myoblasts. Similarly, no differences
were observed in the mRNA ratios for POLG and Tfam on day
5 in myotubes (P ? 0.44 and P ? 0.07, respectively).
In the current research, we have shown that ddI and ddI-
containing regimens are associated with the greatest degree of
mtDNA suppression in HSMCs, most notably in myoblasts.
The patterns of mtRNA decrease were different from those of
mtDNA, particularly for cells treated with NRTI combina-
tions. These findings suggest that evaluating markers beyond
mtDNA can provide additional information on the potential
adverse effects of these drugs on mitochondrial function.
To our knowledge, this is the first study that has investigated
the impact of NRTIs and NRTI combinations on mitochondria
in human skeletal muscle myoblasts, a cell type likely to be
susceptible to mitochondrial damage in vivo. Myoblasts can
differentiate into myotubes (32) and are more abundant in the
skeletal muscle of infants and children than in adults (8, 12).
Moreover, age is known to alter the potential of myoblasts to
differentiate into myotubes (22) and to affect myoblast metab-
olism and proliferation (3). These differences are of particular
interest, because our data indicate that the decline in mtDNA
resulting from ddI exposure was greater for myoblasts than
myotubes. Thus, it is likely that children are more vulnerable
than adults to the mitochondrial toxicity of ddI which could
negatively impact growth and development. Recent in vivo
data also demonstrated that mitochondrial damage at birth in
monkey offspring exposed perinatally to AZT-ddI was severe
and there was no improvement during the first year of life, with
significant reduction of mtDNA in muscle compared to levels
for other NRTI regimens (6). Of note, ddI is the only purine
analog that is commonly used in developing countries, in con-
trast to other NRTIs such as AZT and d4T, which are pyrim-
idine analogs. This is of particular concern in developing coun-
tries, where ddI is widely used as part of the first-line treatment
of HIV-infected children (28, 37).
The decrease in mtDNA abundance is determined by either a
decrease in replication or an increase in degradation of mtDNA.
Important factors for the replication of mtDNA include the nu-
clear genes encoding the mtDNA-specific replication and tran-
scription factors such as POLG and Tfam. In our current study,
the mRNA expression for POLG and Tfam did not demonstrate
any significant changes in HSMCs treated with ddI. It is possible
that a longer observation beyond the 5 days used for our study
might have altered the mRNA expression of POLG or Tfam. It is
pathways, including reactive oxygen species production, uncou-
pools in mitochondria (29). In contrast, the degradation of mito-
chondria by autophagy, specifically called mitophagy (19), plays a
central role in the degradation of whole mitochondria and their
contents (15). Therefore, the effects of each NRTI on mitophagy
may, in part, determine the degree of mtDNA and mtRNA deg-
Interestingly, NRTIs were found to induce different patterns
of decline for mtDNA and mtRNA levels in HSMCs. ddI and
ddI-containing regimens showed a rapid decline in mtDNA
levels but slower and smaller declines in mtRNA. Moreover,
NRTI combinations seemed to lower the mtRNA levels in
HSMCs to a significantly greater extent than single NRTIs,
demonstrating the cumulative negative impact of NRTI com-
binations on mtRNA levels, a finding not observed in mtDNA
levels. Recent data obtained using HepG2 cells also showed
similar negative effects of NRTI combinations on mitochon-
dria (34). These findings, combined with our current data,
suggest the importance of evaluating NRTI combinations and
not relying on single-drug studies if the impact of antiretrovi-
rals on mitochondria is to be assessed in in vitro models (14),
because patients take NRTI combinations as a part of
The decline in mtRNA levels for MTCYB with ddI and
ddI-containing regimens was more pronounced than the de-
cline in MTCO3 mtRNA levels, suggesting a differential impact
of ddI on mitochondrial oxygen phosphorylation (OXPHOS)
complexes. Importantly, MTCYB encodes a subunit of respira-
tory complex III, and MTCO3 encodes subunits of respiratory
complex IV; both transcriptions are initiated by the same
heavy-strand promoter (PH). Therefore, the difference may be
explained by the instability of each mtRNA due to the different
lengths of poly(A) tails at the 3? ends of mtRNA in MTCYB
and MTCO3 (9, 33). Our current data, combined with those of
a previous study using lymphoblast lines (10), suggest the im-
portance of investigating additional markers beyond mtDNA,
such as mtRNA levels, for the evaluation of mitochondrial
toxicity. Further investigations are needed, however, to fully
understand the long-term consequences of these mtDNA and
In agreement with our previous in vivo data (30), mtDNA
significantly increased in myoblasts treated with AZT-contain-
ing regimens. Similar findings are also observed in other in
vitro models, including human hepatoblastoma (HepG2) cells
(5) and HSMCs (2). The mechanisms of increase in mtDNA by
AZT are still unknown; however, these results strongly suggest
that AZT upregulates genes encoding mtDNA. Because AZT
causes significant mitochondrial damage in different in vitro
models (14), the increase in mtDNA may reflect a compensa-
tory response to mitochondrial dysfunction resulting from dif-
ferent causes, including mtDNA POLG (23), oxidative stress
(16), and increases in mtDNA and mitochondrial mass by
oxidative stress (18, 36).
There are limitations of this study. First, we do not know the
actual concentrations of NRTIs in HSMCs, although the drug
levels in culture were designed to approximate those used
clinically. Even so, the intracellular drug concentrations used
for this study may not reflect the concentrations in vivo. One
study using an in vivo rat model demonstrated that the intra-
cellular concentration of ddI in rat hepatocytes was almost half
the extracellular concentration, whereas the intracellular and
extracellular concentrations of AZT were similar (27). Evalu-
ating the assays using concentrations higher than 1? Cmaxmay
be more informative for ddI. Second, although we demon-
strated changes in mtDNA and mtRNA abundance, the actual
functions of mitochondria were not evaluated in the current
study. These include lactate production, mitochondrial mem-
brane potential, and other relevant mitochondrial parameters.
Finally, as noted earlier, the duration of treatment of 2 to 5
days may not reflect the drug effect on mitochondria in patients
receiving long-term antiretroviral therapy.
VOL. 52, 2008NRTIs AND MITOCHONDRIAL TOXICITY IN MUSCLE CELLS2829
In conclusion, we have shown that ddI and ddI-containing
regimens in clinically relevant concentrations have a significant
impact on mtDNA and mtRNA levels in HSMCs, most notably
myoblasts. The results are consistent with our previously pub-
lished in vivo data demonstrating that mtDNA levels in
PBMCs were significantly affected by ddI. These data suggest
that the use of ddI in children should include provider aware-
ness of the potential of mitochondrial toxicity, especially in
developing countries, where ddI has been used widely as a
first-line antiretroviral therapy.
We acknowledge Carol Mundy, Joseph Sanding, and Mary Strauss
at the University of California San Diego for assistance collecting
blood samples from donors and Happy Araneta at the University of
California San Diego for help performing statistical analyses.
We each certify that we have no commercial associations that might
pose a conflict of interests in connection with this article.
This work was supported by the Pediatric AIDS Clinical Trials
Group and by grants from the National Institute of Allergy and Infec-
tious Diseases (5K23AI-56931 to A.S. and AI-39004, AI-27563, AI-
33835, and AI-41110) and grants AI-36214 (Virology Core UCSD
Center for AIDS Research) and AI-32921.
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