ARTÍCULOS ORIGINALES

Acetaldehyde induced developmental and genetic damage in Drosophila melanogaster

Daño genético y del desarrollo inducido por acetaldehido en Drosophila melanogaster

 

Palermo A.M.^1*, Mudry M.D.²

¹ CITEDEF (Instituto de Investigaciones Científicas y Técnicas para la Defensa). J.B. de La Salle 4397 (B1603ALO), Buenos Aires, Argentina.
² GIBE (Grupo de Investigación en Biología Evolutiva), FCEyN-UBA; IEGEBA-CONICET (Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires; Instituto de Ecología, Genética y Evolución de Buenos Aires - Consejo de Investigaciones Científicas y Técnicas). Ciudad Universitaria, Pabellón II, 4º Piso Labs. 43-46. (C1428EHA) Buenos Aires, Argentina.

* Author for correspondence apalermo@citedef.gov.ar

Fecha de recepción: 08/07/2015
Fecha de aceptación de versión final: 26/08/2015

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ABSTRACT

Acetaldehyde (AAld) is a ubiquitous compound in the environment. Exposure may occur in its manufacture or use, in the consumption of alcoholic beverages and through cigarette smoke. It causes distant target effects, including DNA damage, glutathione depletion, enzyme inactivation and cell death. In the present work, its potential teratogenic, mutagenic and recombinagenic effects in somatic cells of D. melanogaster were analyzed. The white/white+ (w/w+) eye mosaic test (Somatic Mutation And Recombination Test, SMART) was applied by mating w/w females with y/Y males. Larvae of 48 ± 4 h were transferred to media with 0.01 %, 0.02 %, 0.05 % and 0.10 % AAld for 12 hs and then moved to regular media until pupation. The emerging w/w+ females were evaluated for malformations in those exposed to the two lower concentrations, and for the presence of white light spots (LS) in the exposed to AAld at the three higher concentrations. A significant increase in the number of malformations was observed in adults born from larvae chronically exposed to AAld. The number of LS that resulted from loss of heterozygosity (LOH) in treated larvae was modified by 0.05 % and 0.10 % AAld that yielded a more than twice rise in the control values of LS/100 eyes (p<0.05). Our findings confirm that AAld is capable by itself of altering the regulation of development and inducing genetic damage in somatic cells of Drosophila melanogaster. These, together with results previously reported in other in vivo and in vitro model systems, highlights the potential role of AAld in the pathogenesis of fetal alcohol syndrome.

Key words: Ethanol; Teratogenesis; Mutagenesis; Loss of heterozygosity; Eye-spot test.

RESUMEN

La exposición a acetaldehído (AAld), un compuesto muy ubicuo en el ambiente, ocurre durante su fabricación y uso, con el consumo de bebidas alcohólicas y/o por el humo de cigarrillos. Causa efectos sobre blancos distantes que incluyen daño al ADN, disminución de glutatión, inactivación enzimática y muerte celular. En este trabajo se estudió su potencial teratogénico y la capacidad de inducir mutagénesis y recombinagénesis en células somáticas de D. melanogaster. Se aplicó la prueba de mosaicismo en ojo white/white+ (w/w+) (Somatic Mutation And Recombination Test, SMART) cruzando hembras w/w con machos y/Y. Larvas de 48 ± 4 h se mantuvieron por 12 hs en medio con acetaldehído 0,01 %; 0,02 %; 0,05 % y 0,10 % (v/v) y luego se dejaron desarrollar hasta adultos en medio normal. Las dos dosis menores se utilizaron para el estudio de malformaciones, mientras que las tres dosis mayores para el estudio de frecuencia de manchas claras (LS) en los ojos de hembras adultas w/w+ Se encontró un aumento estadísticamente significativo en el número de anormalidades en adultos nacidos de larvas expuestas de manera crónica. El número de LS resultantes de la pérdida de heterocigosis (LOH) por los tratamientos de las larvas fue significativo para 0,05 % y 0,10 % (v/v). El AAld produjo un aumento de LS/100 ojos mayor al doble de las frecuencias de control (p<0,05). Estos hallazgos concluyen que el AAld per se es capaz de alterar la regulación del desarrollo y de inducir daño genético en células somáticas de Drosophila melanogaster. Estos resultados, conjuntamente con resultados previos en otros modelos in vivo e in vitro, destacan su rol potencial en la patogénesis del síndrome alcohólico fetal.

Palabras clave: Etanol; Teratogénesis; Mutagénesis; Pérdida de heterocigosis; Eye-spot test.

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INTRODUCTION

Acetaldehyde (AAld) is a ubiquitous compound in the environment. It is an intermediate product of higher plant respiration that is formed as a product of incomplete wood combustion, coffee roasting, burning of tobacco, vehicle exhaust fumes, coal refining and waste processing. The predominant use of AAld is as an intermediate in the synthesis of other chemicals such as perfumes, polyester resins, and basic dyes. It is also used as a fruit and fish preservative, as a flavoring agent, as a denaturant for alcohol, in fuel compositions, for hardening gelatin and as a solvent in the rubber, tanning, and paper industries. Exposure may occur in the manufacture or use of the compound and in the consumption of alcoholic beverages (USEPA, 2000). Besides, cigarette smoke contains abundant reactive aldehydes, including short-chain ones such as AAld and formaldehyde which are relatively long-lived and highly cell permeative molecules, that can cause distant target effects, including DNA damage, glutathione depletion, enzyme inactivation and cell death (Marchitti et al., 2008).
Acute exposure of rats, rabbits and hamsters showed that AAld has low acute toxicity from inhalation and moderate acute toxicity from oral or dermal exposure (USEPA, 1999). Chronic inhalation in hamsters produced changes in the nasal mucosa and trachea, growth retardation, slight anemia, and increased kidney weight. Mammal symptoms of chronic intoxication are similar to those of alcoholism (USEPA, 1999; 2000). In 2012 AAld was designed as “carcinogenic to humans” (Group 1) by the International Agency for Research on Cancer (IARC) and as a consequence renewed attention was brought to the biological effects of this compound (IARC, 2010; 2012). Data from animal studies suggest that AAld may be a potential teratogen however information on its reproductive or developmental effects is scarce. The offspring of rats exposed to AAld by injection showed skeletal malformations, reduced birth weight, and increased postnatal mortality (USEPA, 2000).
In their revision of risk assessment and mechanisms of action of aldehydes, Feron et al. (1991) concluded that AAld is a genotoxic cross-linking agent with limited or no ability to induce gene mutations in bacteria and mammalian cells in vitro, nevertheless it induces chromosomal aberrations and other cytogenetic effects in mammalian cells including human lymphocytes. Recently, Brooks and Zakhari (2014) revised all the biological effects of AAld related to its role as the primary oxidative metabolite of ethanol considering the different AAld-derived DNA and protein adducts that may affect the genome structure and function. In their review they emphasized the role of AAld effects on the mitochondrial genome taking into account that aldehyde dehydrogenase 2 (ALDH2), the primary AAld metabolic enzyme, is located in the mitochondrion. In Drosophila, the detoxification of this aldehyde is also mediated by ALDH (Barbancho et al., 1987). The genotoxic effects reported in the literature indicate that AAld treatments of adult flies resulted in an increased frequency of sex-linked recessive lethals whereas did not affect X chromosomal segregation in oocytes (Woodruff et al., 1985; Vogel, 1992).
Taking into account that studies about developmental effects of AAld are scarce and that, to the best of our knowledge, Drosophila larvae had not been previously used as a model system for the evaluation of this compound, the aim of this work was to investigate the potential teratogenic, mutagenic and recombinagenic effects of AAld in fly larvae.

MATERIALS AND METHODS

The white/white+ (w/w+) eye mosaic test was applied to evaluate the mutagenic and recombinagenic potential of AAld (ethanal, C2H4O, CASRN 75-07-0) in somatic tissues. The principle of the system is the detection of mosaic light spots (LS) in the red eyes of adult females that emerge from exposed larvae that are heterozygous for this marker. LS are the result of “loss of heterozygosity” (LOH) and the consequent expression of the X-linked gene white. (Vogel, 1992; Vogel and Nivard, 1993; 1999; 2000).
Strains of w/w genotype (white females) and y/Y (yellow males) were kindly provided by Dr. Vogel and kept in the fly stocks of the IEGEBA-FCEN-UBA. Larvae of 48 ± 4 h were transferred to media containing AAld at concentrations of 0.01 %, 0.02 %, 0.05 % and 0.10 % (vol/ vol) and kept for 12 hs (chronic treatment), when they were removed and maintained in regular media until eclosion. The toxicity was evaluated by comparing the larval or pupal death in control and treated series taking into account that death larvae appear as dark coloured and that uneclosed pupae are easily distinguished from eclosed ones (Palermo and Mudry, 2011). The eyes of the emerging heterozygous w/w+ females were examined in a solution of 90 % ethanol, 1 % Tween 80 and 9 % water for the presence of white spots (LS) at a magnification of 40–60x. The number of LS was counted in the eyes of females born from larvae exposed to doses 0.02 %, 0.05 % and 0.10 % and control series, and the number of ommatidia affected in each white clone was also recorded. The somatic reversion frequency was estimated by the number of LS per 100 eyes, in order to consider the possible occurrence of more than one spot per eye. The data were evaluated using the ×2 test for proportions, and the multiple decision procedure proposed by Frei and Würgler was applied to diagnose the results as positive, negative, inconclusive, or weakly positive (Frei and Würgler, 1988; 1995). The relative frequencies of LS were compared with the respective negative controls. The occurrence of malformations was studied in the adult flies born from larvae treated with doses 0.01 % and 0.02 %. The results were evaluated by the Kastenbaum and Bowman Test for proportions (Kastenbaum and Bowman, 1970).

RESULTS

None of the concentrations tested (0.01 %, 0.02 %, 0.05 % and 0.10 % v/v) showed differences in larvae or pupal death when compared with control values (about 96-98 % of viability). In our experiments in D. melanogaster we found a rise in the number of malformations in adults born from larvae chronically exposed to AAld (Table 1). The total number of malformations was significantly higher at both concentrations studied (p¡Ü0.05 Kastenbaum and Bowman Test). In control and 0.01 % AAld series only the abdomen is affected, but when the concentration was raised to 0.02 % AAld the abnormalities appeared in abdomen, thorax, wings and eyes.

Table 1. Malformations in D. melanogaster adults born from larvae chronically treated with acetaldehyde (AAld).

Regarding AAld genotoxicity, our results with the eye SMART assay are shown in Table 2. Treatments with 0.02 % AAld did not show statistical differences with control values, while 0.05 % and 0.10 % AAld yielded a significant increase in LS/100 eyes that was more than twice the control values (p<0.05, m=2). Nevertheless, the response was not dosedependent, because 0.05 % and 0.1 % AAld treatments gave similar responses compared to concurrent or historical controls. The distribution of spot sizes is shown in Figure 1. Treatments with 0.02 % AAld that did not increase the frequency of LS present a similar distribution to that of concurrent and historic control series, being size 2 the most frequent class of ommatidia. The treatments with 0.05 % and 0.1 % AAld, that showed significant differences in LS compared to control series, presented a different distribution of spot sizes, where the most frequent size class was 3-4.

Table 2. Frequency of eyes with light spots (LS) after chronic treatment of D. melanogaster larvae with acetaldehyde (AAld).

DISCUSSION

As mentioned in the Introduction, AAld is an active primary metabolic product of ethanol that induces a range of toxic, pharmacological and behavioral responses, due to its ability to elicit cellular and tissue damage (Guo and Ren, 2010). Ethanol is a well-known teratogen, while data about AAld effects on development are scarce. Using frog embryo teratogenesis assay-Xenopus (FETAX) Fort et al. (2003) found that AAld was markedly more potent as a developmental toxicant than ethanol or acetic acid. In rat embryos exposed to AAld Menegola et al. (2001) showed a characteristic embryonic AAld syndrome, histologically characterized by marked cellular death and they proposed that necrosis and apoptosis were the teratological mechanisms responsible of the observed cellular death. In mouse ovary AAld is generated as a by-product during steroidogenesis and can exert toxic effects to impair the differentiation of granulosa cells, reduce ovulation and decrease oocyte quality (Kawai et al., 2012). Ranganathan et al. (1987) using Drosophila for developmental toxicity screening concluded that ethanol itself was responsible for the teratogenic effects observed. However, Langevin et al. (2011) found that AAldmediated DNA damage may critically contribute to the genesis of fetal alcohol syndrome in fetuses, as well as to abnormal development, hematopoietic failure and cancer predisposition in Fanconi anemia patients. Reactive aldehydes are by-products of several metabolic pathways and, without enzymatic catabolism, may accumulate and cause DNA damage. Besides, AAld is able to react with a variety of molecular targets, including DNA and protein, and even to generate an immune response to protein adducts (Romanazzi et al., 2013). These properties justify the hypothesis of being capable by itself, to alter the regulation of the developmental process as it is suggested by the results mentioned above.
Our results are in line with those communicated previously in Drosophila, where chronic feeding with AAld gave weak but reproducible effects for spots in the wing spot assay (SMART) (Graf et al., 1989). Moreover, this compound induced sex linked recessive lethals in fly females (Woodruff, 1985) while it failed to increase X chromosome nondisjunction (Rey et al., 1994). Positive results were also obtained for induction of sister chromatid exchanges (SCE) in human lymphocytes and in mammal cells in vitro (Obe and Ristow, 1977; Obe and Anderson, 1987) for aneuploidy in CHO cells (Dulout and Furnus, 1988) and single strand breaks (SSBs) and double strand breaks (DSBs) in human lymphocytes (Singh and Khan, 1995) AAld raised the frequency of mutation and recombination in Saccharomyces cerevisiae (Albertini et al., 1993). Exposition to AAld has to be considered as an important factor for public health because it could cause an increase in oxidative stress and disease onset, including cancerous transformation processes (Avezov et al., 2014). Kayani and Parry (2010) reported that while ethanol produces in vitro genotoxic effects mainly through an aneugenic mechanism, its metabolite AAld is a clastogen. Kotova et al. (2013) observed that AAld effectively blocks DNA replication elongation in mammalian cells, resulting in DNA double-strand breaks associated with replication. It is interesting to emphasize that these findings are in line with the negative results that we obtained in Drosophila with ethanol using the in vivo eye spot assay of SMART (Palermo et al., 1994) because aneuploid cells are almost inviable in this test system. On the contrary the increased frequency of LS caused by AAld treatments that we report here with the same assay, could occur through a clastogenic mechanism. Our findings confirm that AAld is capable by itself to alter the regulation of development and to induce genetic damage in somatic cells of Drosophila melanogaster, and together with results previously reported in other in vivo and in vitro systems highlights the potential role of AAld in the pathogenesis of fetal alcohol syndrome.
Brooks and Zakhari (2014) considered that the understanding of how AAld impacts genome function in different cells and under different conditions has important implications not only for alcohol-related carcinogenesis, but also for understanding other pathological effects of alcohol as well. Our results in Drosophila intend to contribute to this understanding.

CONFLICTS OF INTEREST

The authors declare that they have no conflicts of interest.

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