Abstract
Abacavir (ABC), zidovudine (AZT), and lamivudine (3TC) are nucleoside analog reverse transcriptase inhibitors (NRTIs) widely used as combination-based antiretroviral therapy against human immunodeficiency virus. Despite effective viral suppression using NRTI combinations, genotoxic potential of NRTIs can be increased when administered in combination. This study investigated the toxic and genotoxic potential of ABC when administered alone or in combination with AZT and/or 3TC using the somatic mutation and recombination test in Drosophila melanogaster. This test simultaneously evaluated two events related to carcinogenic potential: mutation and somatic recombination. The results indicated that ABC was responsible for toxicity when administered alone or in combination with AZT and/or 3TC. In addition, all treatment combinations increased frequencies of mutation and somatic recombination. The combination of AZT/3TC showed the lowest genotoxic activity compared to all combinations with ABC. Therefore, our results indicated that ABC was responsible for a significant portion of genotoxic activity of these combinations. Somatic recombination was the main genetic event observed, ranging from 83.7% to 97.7%.
Keywords
Introduction
Introduction of antiretroviral drugs to treat human immunodeficiency virus (HIV) did not initially result in substantial changes in prognosis for HIV patients. Improvements in prognosis were made with the advent of combination-based antiretroviral therapy (ART), which led to effective HIV suppression, significant immune function recovery, improvement of clinical symptoms, and notably extended patient life span. 1 Nucleoside analog reverse transcriptase inhibitors (NRTIs) are one category of ART medicines and include structural analogs of endogenous nucleosides. These nucleosides are recognized and incorporated erroneously as normal metabolic intermediates during nucleic acid polymerization, which halts viral DNA synthesis. 2
Although antiretroviral drugs are effective in suppressing HIV replication, all classes of antiretroviral agents have multiple side effects. 3 –5 Considering the number of antiretroviral medicines currently in use, the possible number of combinations is large and the choice between these combinations depends strongly on knowledge about toxicity of these medicines. 6 Several studies have reported that NRTIs alone or in combination have demonstrated genotoxic effects when incorporated in host nuclear and mitochondrial DNA such as mutations, micronuclei formation, chromosomal aberrations, sister chromatid exchange, and shortened telomeres, resulting in genomic instability. 7 –9 Genotoxic potential of combinations may be a result of synergistic, antagonistic, or additive effects. 10,11 Furthermore, some NRTI combinations may be more genotoxic than others, influencing efficacy and side effects in long-term use. 9,12,13
Abacavir (ABC) sulfate is an NRTI medicine widely used as a component of ART. ABC was the 15th antiretroviral approved by the United States Food and Drug Administration (FDA) in 1998 for treatment of AIDS in humans. According to the FDA, ABC had no adverse effects on mating performance or fertility of male and female rats at a dose approximately eight times higher than the recommended human dose based on body surface area comparisons. 14 However, few studies have evaluated genotoxicological effects of ABC. Kaushik et al. 15 reported that rats that received ABC for 28 consecutive days had decreased sperm taxa and body weight, suggesting that ABC had toxic potential. Another study demonstrated that therapeutic concentrations of ABC induced numerous DNA double-strand breaks (DSBs) in chromosomal DNA. 16 Thus, we aimed to investigate the overall toxic and genotoxic potential of ABC when administered alone or in combination with the NRTIs zidovudine (AZT) and/or lamivudine (3TC; Figure 1) using the somatic mutation and recombination test (SMART) in Drosophila melanogaster.
Molecular structures of NRTIs: (a) ABC initials, (b) AZT, and (c) 3TC. NRTI: nucleoside analog reverse transcriptase inhibitor; ABC: abacavir sulfate; AZT: zidovudine; 3TC: lamivudine.
Materials and methods
Chemicals
Abacavir sulfate ((1 S,4 R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol sulfate; CAS no. 188062-50-2), AZT (1-((2 R,4 S,5 S)-4-azido-5-(hydroxymethyl)oxolan-2-yl)-5-methylpyrimidine-2,4-dione; CAS no. 30516-87-1), and 3TC (4-amino-1-((2 R,5 S)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl)pyrimidin-2-one; CAS no. 134678-17-4) (Figure 1) were obtained from Indústria Química de Goiás (Goiânia, Brazil) and Hospital de Doenças Tropicais (Goiânia, Brazil).
Strains and cross
Two strains of D. melanogaster were used: (i) mwh/mwh (multiple wing hairs) and (ii) flare-3 (flr3/In(3LR)TM3, ri ppsep I(3)89Aa bx34eBds). Expression of the mwh marker is involved in the formation of multiple hairs in each cell, while the flr3 marker (flare-3 strain) leads to the formation of mutant hairs that resemble a candle flame. 13 Flies were maintained in vials containing banana-based culture medium (1000-ml water, 12-g agar, 800-g banana, 15-g biological yeast, and 2-g Nipagin) and incubated in a BOD-type chamber (model: SL224; SOLAB – Equipamentos para Laboratórios Ltda, São Paulo, Brazil) at 25 ± 1°C and 65% relative humidity.
A standard cross (ST) was performed using mwh/mwh males and virgin flare-3 females, which produced descendants with two genotypes: trans-heterozygous (mwh/flr3 ) and balanced heterozygous (mwh/TM3). The mwh/flr3 progeny allowed for identification of mutagenic and recombinogenic events and the mwh/TM3 progeny revealed only mutations, since the TM3 chromosome has multiple inversions that block recombination. 17,18
Toxicity
To test toxicity, groups of 100 third-stage D. melanogaster larvae from ST cross were treated for approximately 48 h with different concentrations of ABC alone or in combination with AZT and/or 3TC. The ABC concentrations used were 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, and 16.0 mg/ml. For combination treatments, the following proportions of cocktails from clinical protocols were used: 1:1 (ABC/AZT), 2:1 (ABC/3TC and AZT/3TC), and 2:1:2 (ABC/3TC/AZT). Surviving adult flies were counted to establish survival rates. Results were obtained from two independent experiments.
SMART in somatic cells of D. melanogaster
Third-stage D. melanogaster larvae obtained from ST cross were placed in tubes containing 0.9 g of instant mashed potatoes (Hikari® brand, São Paulo, Brazil) culture medium, hydrated with 3 ml of the treatment solutions. The four lowest concentrations of ABC (0.25, 0.5, 1.0, and 2.0 mg/ml) were used, all of which resulted in survival rates greater than 70%. Distilled water was used as the negative control. Larvae were exposed to treatments with ABC alone or in combination (AZT and/or 3TC) until the pupal stage, as previously described in the toxicity evaluation.
After pupae eclosion, adults were collected and preserved in 70% ethanol. The wings were removed, placed on glass slides, and analyzed using an optical light microscope with a 40× objective lens. A total of 40 individual adults were analyzed for each tested concentration, derived from two independent experiments. For statistical analyses, frequencies of three types of spot (small single, large single, and twins) and total spots resulting from different concentrations of ABC alone or in combination (AZT and/or 3TC) were compared to the respective negative controls. Statistical significance was determined using the conditional binomial test of Kastenbaum and Bowman, 19 following the multiple decision test proposed by Frei and Würgler, 20 resulting in four possible results: positive, negative, inconclusive, and weakly positive. To calculate the percentage of recombination and mutation, the ratio between the total of spots induced in mwh/flr3 and mwh/TM3 offspring was used, according to Frei and Würgler. 20
Results
Toxicity
Toxic effects of ABC alone and in combination with AZT and/or 3TC were evaluated and the number of surviving flies per treatment provided data to establish survival rates (Figure 2). The results showed that ABC alone interrupted larval development at concentrations higher than 2 mg/ml. Combination treatments including ABC resulted in toxicity similar to ABC alone. Thus, the presence of AZT, 3TC, or AZT + 3TC did not significantly alter toxicity induced by ABC, suggesting that ABC was responsible for increased toxicity.
Survival rates of Drosophila melanogaster progeny resulting from ST. Larvae were exposed to different concentrations of ABC alone (a), ABC/3TC (b), ABC/AZT (c), ABC/3TC/AZT (d), and AZT/3TC (e). In combinations, the following proportions of cocktails derived from clinical protocols were used: 1:1 (ABC/AZT), 2:1 (ABC/3TC and AZT/3TC), and 2:1:2 (ABC/3TC/AZT). ST: standard; ABC: abacavir; AZT: zidovudine; 3TC: lamivudine.
Genotoxicity
In all treatments, mwh/flr3 offspring had a greater frequency of mutant spots compared to negative controls (Table 1). The frequencies of mutant spots induced by the combination AZT/3TC were lower than those observed in all treatments with ABC. In addition, excluding the highest concentration of the ABC/3TC/AZT combination tested, the combination of ABC/3TC induced the highest rate of mutant spots (Table 1). The offspring of mwh/TM3 also had significantly greater total frequency of spots compared to the negative control, except the treatment with AZT/3TC in which spot frequency results were inconclusive compared to the negative control (Table 1).
Results obtained with the SMART in Drosophila melanogaster wing cells, with trans-heterozygous (mwh/flr3 ) and balanced heterozygous descendants (mwh/TM3) of the ST, treated with ABC initials, AZT, and/or 3TC.
SMART: somatic mutation and recombination test; ST: standard cross; ABC: abacavir sulfate; AZT: zidovudine; 3TC: lamivudine; MH: trans-heterozygous flies (mwh/flr3); BH: balancer-heterozygous flies (mwh/TM3).
a Statistical diagnosis according to Frei and Würgler (1988): U-test, two sided; probability levels: +, positive; -, negative; i, inconclusive. p ≤ 0.05 vs. negative control.
b Including rare flr3 single spots.
c Considering mwh clones from mwh single and twin spots.
d Frequency of clone formation: clones/flies/48,800 cells (without size correction).
e C = 48.000, i.e., approach number of cells examined for individual.
fCalculated as: Recombination = 100 - mutation. Mutation = frequency of clones in BH flies/frequency of clones in MH flies × 100.
g Balancer chromosome TM3 does not carry the flr3 mutation and recombination is suppressed, due to the multiply inverted region in this chromosome.
Using the results obtained from genotoxic action of mwh/flr3 and mwh/TM3 offspring, we quantified the contribution of both mutation and recombination events. The recombinogenic ratios ranged from 83.7% to 97.7%, indicating that somatic recombination was the major genetic event induced in all treatments.
Discussion
The evolution of combined drug treatment against HIV is one of the most remarkable medical advancements of the 21st century. Since the initial approval of AZT by the FDA in 1987, the number of drugs used to treat HIV has grown rapidly and turned a fatal disease into a chronic illness. 5 Therefore, maximizing the safety and tolerability of ART is a high priority, since HIV patients must take antiretroviral agents for decades. 21 Based on the importance of ART in HIV treatment, investigation of genotoxic potential of drugs used in HIV therapy is paramount. In this study, we analyzed the toxic and genotoxic potential of ABC alone or in combination with AZT and/or 3TC in somatic cells of D. melanogaster.
First, we investigated toxicity of ABC alone or in combination with AZT and/or 3TC in D. melanogaster larvae. The results showed that combinations containing ABC had toxic effects similar to ABC alone. These data corroborate a previous study, showing that cytotoxic effects induced by the combination ABC/3TC/AZT were similar to the effects induced by ABC alone in TK6 human lymphoblastoid cells. 10 Moreover, a previous study showed decreased sperm count and testis weight in rats chronically treated with ABC, suggesting induction of male infertility in animals. 15 Therefore, these results indicated substantial ABC-induced cytotoxicity.
ABC induced a concentration-dependent increase in the number of mutant spots in D. melanogaster. In previous studies that investigated genotoxicity of ABC alone, ABC was not mutagenic in bacterial mutagenicity assays. 22 However, other studies reported that ABC induced chromosomal abnormalities in human lymphocytes and in rats and induced micronuclei in bone marrow and in peripheral blood cells of treated rats and mice. 15,23 Moreover, orally administered ABC increased the incidence of malignant and nonmalignant tumors in rats and mice, indicating that this drug is a potent rodent carcinogen. 23,24
Genotoxic evaluation of combinations of ABC with AZT and/or 3TC in D. melanogaster showed that the total frequency of mutant spots induced by ABC when administered alone was lower than the total frequency observed in combinations containing ABC. Comparison of ABC alone with ABC/3TC, ABC/AZT, ABC/3TC/AZT, and AZT/3TC demonstrated that a combination without ABC (AZT/3TC) resulted in the lowest genotoxic action. Similar results were observed in patas monkeys, since animals exposed to the ABC/3TC/AZT combination presented more cells containing micronuclei than those treated with the AZT/3TC combination. 9 These results suggested that AZT and 3TC potentiated the genotoxic effect of ABC, and ABC alone was more genotoxic than the combination of AZT and 3TC. This may be a result of (i) impaired DNA repair, leading to uncorrected DSBs promoted by ABC in D. melanogaster DNA; (ii) recombinogenic potentiation when using a drug cocktail; (iii) increased DNA incorporation of nucleoside analogs when drugs are used in combination; and/or (iv) an imbalance in the natural deoxynucleotide pool, leading to genomic instability and increased risk of non-repairable or permanent damage to nuclear and mitochondrial DNA. 11,16,25,26 Moreover, the combination ABC/3TC showed greater genotoxic potential than ABC/AZT. These data suggested that 3TC was more genotoxic in the presence of ABC than AZT, but that ABC was primarily responsible for genotoxic action of these combinations.
Our results showed that genotoxicity observed in D. melanogaster was mostly due to recombination events (83.7–97.7%) rather than mutations. NRTIs appear to target cellular factors involved in interphase organization of constitutive heterochromatin, important for chromosome stability, and segregation. 27 This outcome is important, since recombinogenic activity is responsible for the loss of heterozygosity. If this event occurs in patient’s cells, recombination caused by ART may promote manifestation of hereditary recessive diseases or may be involved in progression of neoplasias. 18
Conclusions
In summary, our study highlights the importance of establishing mutagenic and recombinogenic profiles of medicines used in ART, due to the long-term nature of this treatment strategy. Therefore, it is crucial to analyze these medicines alone and in combination in different cellular models to identify their toxic and genotoxic activities to ensure the quality of life of patients.
Footnotes
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The author Cláudia de Jesus Silva Carvalho was supported with a scholarship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil, and the author Elisa Flávia Luiz Cardoso Bailão was supported by Universidade Estadual de Goiás with a fellowship of the program PROBIP (Scientific Production Support Program). This work was supported by Fundação de Amparo à Pesquisa do Estado de Goiás.
