Genetica 95: 103-109, 1995
THE TOXICITY OF AZIDOTHYMIDINE (AZT) ON HUMAN AND ANIMAL
CELLS IN CULTURE AT CONCENTRATIONS USED FOR ANTIVIRAL THERAPY
David T. Chiu & Peter H. Duesberg
Dept. of Molecular and Cell Biology, Stanley Hall, University
of California at Berkeley, Berkeley, CA 94720, USA
AZT, a chain terminator of DNA synthesis originally developed for chemotherapy,
is now prescribed as an anti-human immunodeficiency virus (HIV) drug at
500 to 1500 mg/person/day, which corresponds to 20 to 60 µM AZT.
The human dosage is based on a study by the manifacturer of the drug and
their collaborators, which reported in 1986 that the inhibitory dose for
HIV replication was 0.05 to 0.5 µM AZT and that for human T-cells
was 2000 to 20.000 times higher, i.e. 1000 µM AZT. This suggested
that HIV could be safely inhibited in humans at 20 to 60 µM AZT.
However, after the licensing of AZT as an anti-HIV drug, several independent
studies reported 20 to 1000-fold lower inhibitory doses of AZT for human
and animal cells than did the manufacturer's study, ranging from 1 to 50
µM. In accord with this, life threatening toxic effects were reported
in humans treated with AZT at 20 to 60 µM. Therefore, we have re-examined
the growth inhibitory doses of AZT for the human CEM T-cell line and several
other human and animal cells. It was found that at 10 µM and 25 µM
AZT, all cells are inhibited at least 50% after 6 to 12 days, and between
20 to 100% after 38 to 48 days. Unexpectedly, variants of all cell types
emerged over time that were partially resistant to AZT. It is concluded
that AZT, at the dosage prescribed as an anti-HIV drug, is highly toxic
to human cells.
AZT (3'-azido-3'-deoxythymidine) is an analog of thymidine in
which the 3' hydroxyl group is replaced by an azido group. This
prevents the extension of a growing DNA strand ending with AZT to the
five prime end of another nucleotide triphosphate. Thus AZT functions
as a chain terminator of DNA synthesis.
AZT was originally designed in the 1960s to be used as
chemotherapy for leukemia (Horwitz, Chua & Noel, 1964). The
rationale for cancer chemotherapy is to kill cancer cells during
mitosis with cytotoxic chemicals like AZT. Because chemicals cannot
distinguish cancer cells from normal cells, the price for
chemotherapy is the death of normal cells that are in mitosis.
Therefore, chemotherapy must be restrictod to days or weeks.
Successful chemotherapy kills the cancer before it kills the host.
Since 1987, chronic administration of AZT and similar nucleoside
analogs, like ddC and ddI, have been prescribed to AIDS patients to
inhibit human immunodeficiency virus (HIV), the presumed cause of
AIDS (Fischl et al., 1987; Richman et al., 1987; Yarchoan et al.,
1991). Since 1990, AZT has also been prescribed to healthy HIV
antibody-positive persons to prevent AIDS (Volberding et al., 1990;
Tokars et al., 1993; Seligmann et al., 1994). The rationale is to
inhibit HIV DNA synthesis at doses that do not inhibit cell DNA
synthesis (Yarchoan et al., 1991). It is claimed by
Burroughs-Wellcome, the manufacturer of AZT, and its collaborators
that this can be achieved, because AZT would inhibit DNA synthesis
with HIV DNA polymerase in vitro 100 times more effectively than DNA
synthesis with cellular DNA polymerase (Furman et al., 1986).
Moreover, this study claimed that in vivo AZT was 2000 to 20,000
times more inhibitory to HIV replication, i.e. at 0.05 to 0.5 /1M,
than to cell division, i.e. at 1000 µM (Furman et al., 1986).
Accordingly, anti-HIV doses of AZT were chosen to fall into this
therapeutic window, e.g. to be 500 to 1500 mg per person per day, or
about 20 to 60 µM per kg per day (Furman et al., 1986; Fischl et al.,
1987; Volberding et al., 1990; Physicians' Desk Reference, 1994).
However, in view of its inherent cytotoxicity, AZT has been
questioned as an acceptable anti-HIV drug on three theoretical
grounds (Duesberg, 1992):
(i) Even if AZT were to inhibit HIV DNA synthesis 100 times more
than cell DNA synthesis, it could not 'selectively' inhibit HIV, as
is claimed by the manufacturer (Furman et al., 1986). Since HIV DNA
measures only 10 kb and cell DNA measures 106 kb, and since both DNAs
are made in vivo simultancously inside the same cell, cell DNA
provides a 105-fold bigger DNA target for AZT toxicity than does HIV
DNA. Therefore, the 100-fold higher selectivity of AZT claimed for
HIV DNA synthesis is immaterial.
(ii) Inhibition of HIV DNA synthesis in HIVantibody positive
persons is completely unnecessary, because HIV does not spread in tte
presence of antiviral antibody (Duesberg, 1992). It is for this
reason that only about 1 in 1000 T-cells is ever infected in
HIV-antibody positive persons (Duesberg, 1992). The fact that only
about 0.1% of all susceptible T-cells are ever infected by HIV in
HIV-positive persons with and without AIDS proves that HIV is very
effectively neutralized by antiviral immunity. Moreover, there is no
correlation between the number of HIV-infected cells and AIDS
(Duesberg, 1993; Piatak et al., 1993). For example, there are healthy
HIV-positive persons who have 30 to 40 times more HIV-infected cells
than AIDS patients (Simmonds et al., 1990; Bagasra et al., 1992; Duesberg, 1992).
(iii) Since only about 1 in lOOO T-cells are ever infected by HIV
in persons with or without AIDS (Duesberg, 1992; 1992), AZT must kill
999 uninfected cells in order to kill just one HIV-infected cell - a
very poor pharmacological index.
Thus theory predicts that AZT cannot selectively restrict HIV
replication in vivo. AZT can only inhibit HIV by killing infectod and
uninfected target cells. Theory further predicts that AZT is
unacceptable as anti-HIV therapy in HIV-antibody positive persons,
because it will kill 999 uninfected cells for every infected cell.
In response to these theoretical considerations it is argued by
the manufacturer of AZT and its collaborators that, contrary to
expectations, AZT is an effective anti-HIV drug, because cell
division was observed to be 2000 to 20,000 times more drug-resistant
than HIV replication (Furman et al., 1986).
However, after AZT had been licensed for human use, several
independent studies reported that the drug is about 20 to 1000 times
more toxic to human cells in culture than the manufacturer had
claimed, i.e. that the half inhibitory doses (ID 50) ranged between 1
and 50 µM (Table 1). In accordance with these results, life
threatening toxicity including anemia, leukopenia, nausea, muscle
atrophy, dementia, hepatitis and mortality, has been documented in
humans treated with 20 to 60 µM AZT (Mir & Costello, 1988; Duesberg,
1992; Freiman et al., 1993; Tokars et al., 1993; Bacellar et al.,
1994; Goodert et al., 1994; Seligmann et al., 1994). If these results
were correct, both the dosage of AZT prescribed to humans and the
advisability of AZT as an anti-HIV drug need to be reconsidered.
In view of up to a 1000-fold discrepancy between the cytotoxicity
of AZT reported by the manufacturer and his collaborators (Furman et
al., 1986) and the cytotoxicitiesreported by other investigators
(Table 1), we set out to redetermine the cytotoxicity of AZT. We have
investigated the effects of AZT on the human CEM T-cell line and on
several other human and animal cells in culture. In contrast to the
previous studies, that measured toxicity over 1 to 3 rounds of
mitoses, we decided to measure long-term toxicity over several weeks,
representing up to 24 consecutive cell divisions. We reasoned that
this experimental design would more closely mimic human exposure,
which is indefinite, extending over numerous mitoses (Fischl et al.,
1987; Volberding et al., 1990; Physicians' Desk Reference, 1994).
Under the conditions AZT is prescribed as an anti-HIV drug, i.e.
chronic application, it could indeed be more toxic than it is after
only one or a few mitoses studied earlier, because non-lethal
mutations would accumulate in surviving cells. Our experimental
design would detect cumulative mutational toxicity acquired over
several mitoses, in addition to the complete cytotoxicity observed in
one or a few mitoses.
Materials and methods
Materials. RPMI 1640 medium, Dulbecco's Modified Eaglets medium,
and fetal bovine serum were purchased from Gibco Laboratories (Grand
Island, NY). Serum Plus was purchased from JRH Biosciences (Lenexa,
KS). AZT was purchased from Sigma Chemical Co. (St. Louis, MO).
ID50 at µM AZT
(Furman et al., 1986)
human T-cell, line H9
(Balzarini, Herdewijn & De Clercq, 1989)
human T-cell, line CEM
(Mansuri et al., 1990)
human T-cell, line CEM
(Lemaître et al., 1990)
humanT-cell, line CEM
(Avramis et al., 1989)
human T-cell, line CEM
(Sommadossi et al., 1990)
human bone marrow
human bone marrow
(Inoue et al., 1989)
human bone marrow
human bone marrow
(Mansuri et al., 1990)
mouse bone marow
(Gogu, Beckman & Agrawal, 1989)
mouse bone marrow
mouse fetal liver
Table 1. 50% inhibitory dose of AZT for human and animal cells as reported by various laboratories.
Culture conditions of cells grown in suspension. The CEM human
Iymphoid T-cell line was provided by Robert F. Garry, Tulane
University School of Medicine, New Orleans, LA. CEM T-cells were
suspended in 5 mL of RPMI 1640 medium enriched with 10% Serum Plus in tissue culture flasks (25 cm2 growth area, Falcon) and were
propagated at 37°C in humidified air with 6.5% CO2. The CEM
T-cells were maintained at a density around 3 x 105 cells per mL by diluting them 1:2 every other day. The medium was changed every day
by spinning down the cells for 5 min in a clinical centrifuge at 6000
rpm and then resuspending them in fresh medium. AZT was added twice
every day at 10 and 25 µM concentrations by micropipets with sterile
tips. AZT additions were made at about 12 h intervals. A control
flask of CEM T-cells was passaged identically without the addition of
AZT. A 10 /1L aliquot of evenly distributed cells was counted every
other day with a hematocytometer.
Culture conditions of cells grown attached to Petri dishes. The
C3H mouse fibroblast cell line, the Hs-27 human foreskin cell line
and the WI-38 human lung cell line were purchased from the American
Type Culture Collection. The secondary Chinese Hamster lung cells
were prepared from animals in our lab. Each of these cell types was
cultured while attached to Petri dishes (100 x 20 mm, Falcon) in 10
mL of Dulbeccots Mod ified Eagle's medium enriched with 10% fetal
bovine serum at 37°C in humidified air with 6.5% CO2. Each of
the monolayer cell types was sceded of approximateIy I X 105 cells on
a 10-cm dish containing 10 mL of medium. The medium in each dish was
changed every day. AZT additions were also made twice a day at 10 and
25 µM concentrations. The cells were countod with a Coulter counter
by placing a 200 pL sample of evenly distributed cells in 10 mL of
isotonic buffered saline solution. Each AZT-treated culture was split
1:5 when the control dish had reached 100% confluency.
Fig. 1. The effect of AZT, at 10 µM and 25 µM, on the growth rate of the human CEM T-cell line maintained as described in the text.
The effect of long-term AZT treatment on the viability of the
human CEM T-cell line. To determine the cytotoxicity of AZT on the
human CEM T-cell line in culture, parallel cultures were incubated
with 10 µM, 25 µM AZT and without AZT (see Materials and methods). The
untreated cells were maintained at saturation density of CEM cells,
which is about 3 x 105 cells per mL in our conditions. Each culture
was divided 2-fold every 48 h, by which time the AZT-free control had
regained saturation density.
As can be seen in Fig. 1, after four days the cell count of the
culture at 25 µM AZT had been reduced to half of the control, and
that of the culture at 10 µM AZT to two thirds of the control. After
12 days the cell densities of both AZT-treated cultures had been
reduced to a third of the control culture. From then on, the density
of the culture at 25 µM AZT continued to decline at a decreasing
rate, and that of the culture at 10 µM AZT stabilized (Fig. 1).
One possible explanation of the decreasing sensitivity of
surviving CEM cells to AZT over time is that the dividing portion of
the cells takes up all AZT in a short time, and that the resting
portion of cells subsequently enters mitosis in a culture depletod of
AZT. Another explanation suggests that variants are selected that do
not incorporate AZT into DNA. To distinguish between these
possibilities each AZT-treated culture was further divided into two.
One of the two subcultures was maintained with daily medium changes
containing 10 and 25 µM AZT respectively as before. The other
subculture was supplemented, 12 hours after the medium including AZT
had been changed, with the equivalent of an extra 10 and 25 µM AZT
respectively. All cultures were further incubated under these
conditions for another 32 to 36 days.
It can be seen in Fig. I that even at two daily applications of
AZT at 10 µM, a decreasing fraction of T-cells retained viability for
14 days (when the culture became contaminated). However, no survivors
were observed after 14 days at two daily applications of 25 µM AZT.
It is concluded that T-cell variants are selected, on long-term
exposure to AZT, that are relatively resistant to AZT compared to the
average T-cell prior to treatment.
The effect of long-term exposure to AZT on the viability of human
and animal fibroblasts. To determine whether other human and animal
cells are similar to human T-cells with regard to AZT-sensitivity,
the viability of a human lung (WI) and foreskin (Hs) cell line, of a
mouse cell line (C3H) and of secondary Chinese hamster cells (C.H.)
was studied in AZT.
Each of the different cell types was seeded at 1 x 105 cells per
10 cm dish and exposed to AZT at 10 and 25 µM (see Materials and
methods). AZT was added to each dish twice every day, once in the
morning and again at night as described above. The inhibition of cell
growth was expressed as the percentage of cells in the AZT culture
compared to that of the untreated control. The cells were counted by
the time the control had reached confluency (Fig. 2). The first count
of cells was taken at the end of two weeks when all control dishes
had become completely confluent. Thereafter control cells were split
1:4 and allowed to reach confluency again. This process was repeated
several times as shown in Fig. 2.
As can be seen in Fig. 2, the general pattern of AZT-sensitivity
observed with T-cells was confirmed with other human and animal
cells. C3H mouse cells appeared to be most sensitive to the effects
of AZT. At day 14, the densities of C3H cells, maintained at both
concentrations of AZT, had already declined to below 50 percent of
the control. Possibly due to a counting error, the density of C3H
cells at 10 µM AZT appeared lower than that of cells at 25 µM AZT.
After the same time, the concentrations of Hs-27, WI-38, and C.H.
cells ranged from 50 to 60 percent of the control at 25 µM AZT, and
from 60 to 70 percent of the control at 10 µM AZT.
From 14 to 38 days of AZT treatment all fibroblast cells remained
at about half the density of the controls. However, the densities of
C3H and Hs-27 cell lines gradually increased over time at both
concentrations of AZT. By day 38, the density of Hs-27 cells at both
AZT concentrations had reached up to 80 percent of the control.
Fig. 2. The effect of AZT, at 10 µM and 25 µM, on the growth of
human lung (Wl), and foreskin cells (Hs), on mouse fibroblasts (C3H)
and on secondary Chinese hamster (C.H.) fibroblasts. AZT-treated
cells were counted whenever the untreated control culture had reached
(i) AZT toxic to human cells in the micromolar range. Our results
indicate that long-term exposure to AZT inhibits the growth of human
CEM T-cells about 50% at 10 µM, and gradually up to 100% at 25 µM.
Similar results were obtained with human lung and foreskin cells, and
also with mouse and Chinese hamster cells, although complete
inhibition was not observed with any of these cells under our
conditions. Thus our results confirm and extend those of others
summarized in Table 1, that AZTis toxic to human cells in the
micromolar range. Indeed AZT, like all other nucleotide analogs of
DNA,is expectod to be toxic in the micromolar range, because the
Michaelis constants of authentic nucleotide triphosphates are also in
the micromolar range (Kornberg, 1980).
These results are incompatible with the claim of the manufacturer
and its collaborators that AZT is only toxic to human cells in the
millimolar range. That claim is also hard to reconcile with the
manufacturer's own observation that HIV replication is inhibited by
AZT at 0.05 to 0.5 µM (Furman et al., 1986). Since (i) HIV and cell
DNA are both replicated in vivo inside the same cellular vesicle and
at the same time (Rubin & Temin, 1958; Weiss et al., 1985), (ii)
retroviral and cellular DNA synthesis depend on the same
triphosphatepools, and (iii) retroviral DNAis a 105-fold smaller
target for AZT than cell DNA, HIV DNA synthesis cannot be more
sensitive to AZT than cell DNA. In fact target theory predicts the
Thus the preponderance of evidence casts doubts on the claim of
the manufacturer of AZT and its collaborators that AZTis only toxic
to cells in the millimolar range (Furman et al., 1986).
(ii) Resistance of human and animal cells to longterm exposure to
AZT. Unexpectedly, partially AZT resistant variants emerged from human
T-cells and all other cells testod on continued exposure to AZT at 10
to 25 µM for 38 to 48 days. These variants did not reach the densities
of untreated control cells, but continued to divide in the presence
of AZT at various rates. Further work is needed to analyze the basis
for the relative AZT-resistance acquired by human and animal cells
upon long-term exposure to AZT.
(iii) Toxicity of AZTat micromolar concentrations calls for
reoppraisal of its use as an anti-HlV drug. The cell culture results
described by us and others predict that AZT is toxic to humans at the
20 to 60-micromolar level, the concentrations at which it is
prescribed as an anti-HIV drug. Even though our results show that
human and animal cells acquire some resistance against AZT upon
long-term exposure, no cell has achieved complete resistance to AZT
under the conditions tested. This prediction is confirmed by numerous
clinical studies that describe life threatening toxic effects in
humans treatod with AZT at 20 to 60 µM (see Introduction). Thus our
data and those of others call into question the merits of AZT as an
anti-HIV drug, particularly at the doses currently prescribed to
We thank Robert F. Garry, Tulane Univ. New Orleans, for the human
CEM T-cell line and for generous advice, and Gedge D. Rosson, UC
Berkeley, for preliminary results and discussions. This investigation
was supportod in part by the Council for Tobacco Research, USA, and
private donations from Thomas Boulger (Redondo Beach, Calif., USA),
Glenn Braswell (Los Angeles, Calif., USA), Dr. Richard Fischer
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