Effects of differential alcohol dehydrogenase activity on the developmental toxicity of ethanol in Drosophila melanogaster

The role of alcohol dehydrogenase (ADH) activity in ethanol toxicity was investigated in Drosophila melanogaster. Flies from three congenic Adh strains (high, medium, and low ADH activity) were allowed to deposit eggs on medium containing 0, 4, or 8% ethanol. The resulting larvae were allowed to complete their development in the medium, and emerging flies were examined for defects. Flies with high ADH activity had malformation incidences of 0.8, 2.4, and 5.2% at 0, 4, and 8% ethanol, respectively. The comparable incidences for the low ADH strain were 1.0, 4.1, and 8.4%, while those for the medium ADH strain were intermediate in value. These results indicate that ethanol teratogenesis may be inversely related to ADH activity. When larvae were treated with ethanol for different lengths of time during development, the incidence of defects in flies from the high ADH strain was 3.9% when exposure started at the first instar and 3.09% when exposure started at the third instar. Results of the same exposures for the intermediate ADH strain were 5.2 and 3.4%, respectively, while those for the low ADH strain were 6.9 and 5.5%, respectively. Thus, length of ethanol exposure was directly related to the increased incidence of malformations in all tested Drosophila strains. For all tested strains, defect incidences appeared to be dose-related as well, regardless of length of exposure. ADH in Drosophila has a dual function and thus can catalyze oxidation of both ethanol and its toxic metabolite, acetaldehyde. This suggests that ethanol is the proximate teratogen in Drosophila.     

Teratogenic evaluation of metronidazole and ornidazole using Drosophila melanogaster as an experimental model

BACKGROUND: Drosophila and vertebrates show similarities that suggest that the mechanisms involved in the induction of developmental defects may be similar in both. Therefore, Drosophila has been proposed as a useful, rapid, and economical model in the preliminary screening for teratology studies. The objective of the present study was to investigate the effect of metronidazole (MTZ) and ornidazole (ONZ) on the developmental stages of Drosophila melanogaster. METHODS: Samarkand wild-type females were allowed to lay eggs for 24 hr in media containing MTZ or ONZ at concentrations of 0, 500, 1000, and 2000 microg/ml. When larvae completed their development, the emerging flies were counted and examined for morphological abnormalities. RESULTS: After the analysis of 400-1000 flies for each concentration, ONZ-treated flies did not show an incidence of malformations above control values, although a significant high number of individuals with reduced body size was observed (p < 0.005, chi2 test). On the other hand, the 1000- and 2000-microg/ml MTZ-treated series presented higher frequencies of total abnormalities than did concurrent and historic controls (p < 0.05, chi2 test), indicating an MTZ effect during developmental morphogenesis. CONCLUSIONS: These findings contribute to the characterization of both nitroimidazoles, which are widely used, especially in underdeveloped countries. At the same time, this Drosophila bioassay is sensitive enough to detect differential effects of MTZ and ONZ (abnormalities vs. growth effects), showing specificity and selectivity.     

Sodium nitroprusside toxicity in Drosophila melanogaster: delayed pupation, reduced adult emergence, and induced oxidative/nitrosative stress in eclosed flies

The toxicity of sodium nitroprusside (SNP) was tested on the Drosophila melanogaster model system. Fly larvae were raised on food supplemented with SNP at concentrations of 0.01-1.5 mM. Food supplementation with SNP caused a developmental delay in flies and reduced adult eclosion. Biochemical analyses such as levels of oxidative stress markers and activities of antioxidant and associated enzymes were carried out on 2-day-old flies emerged from control and SNP-fed larvae. Larval exposure to SNP resulted in lower activities of aconitase and catalase in adult flies relative to the control cohort. However, larval treatment with SNP led to higher carbonyl protein content and higher activities of superoxide dismutase, glucose-6-phosphate dehydrogenase, thioredoxin reductase, and glutathione-S-transferase in flies. Among the parameters tested, aconitase activity and developmental end points may be useful early indicators of toxicity caused by SNP. The study also suggests that the toxicity of SNP may arise not just from its direct effects, but also from its decomposition products such as nitric oxide and iron ions.     

S-nitrosoglutathione-induced toxicity in Drosophila melanogaster: Delayed pupation and induced mild oxidative/nitrosative stress in eclosed flies

The toxicity of the nitric oxide donor S-nitrosoglutathione (GSNO) was tested on the Drosophila melanogaster model system. Fly larvae were raised on food supplemented with GSNO at concentrations of 1.0, 1.5 or 4.0 mM. Food supplementation with GSNO caused a developmental delay in the flies. Biochemical analyses of oxidative stress markers and activities of antioxidant and associated enzymes were carried out on 2-day-old flies that emerged from control larvae and larvae fed on food supplemented with GSNO. Larval exposure to GSNO resulted in lower activities of aconitase in both sexes and also lower activities of catalase and isocitrate dehydrogenase in adult males relative to the control cohort. Larval treatment with GSNO resulted in higher carbonyl protein content and higher activities of glucose-6-phosphate dehydrogenase in males and higher activities of superoxide dismutase and glutathione-S-transferase in both sexes. Among the parameters tested, aconitase activity and developmental end points may be useful early indicators of toxicity caused by GSNO.     

Nondisjunction induced by ethanol in Drosophila melanogaster females.

The effect of ethanol on chromosomal segregation was investigated in Drosophila melanogaster females homozygous for a structurally normal X chromosome marked with the recessive mutation yellow (y/y). For chronic treatments the females were kept from eclosion in food supplemented with 10% or 15% (v/v) ethanol, mated 24 or 48 h later to wild-type males and brooded in freshly prepared ethanol food. For the acute treatments 24- or 48-h-old females were exposed for 60 min to a 75% (v/v) ethanol solution by means of soaked tissue paper placed at the bottom of regular culture vials and brooded daily after mating. The results obtained show that: (1) both treatments significantly increased the frequency of X-chromosome nondisjunction; (2) after acute treatment this effect declined in later broods; (3) the yield of malformed flies in the progeny of acutely treated females was significantly higher than control values and also declined in later broods; (4) ovary analysis showed that chronic ethanol treatments caused a cessation of egg production. The induction pattern of nondisjunction and malformed flies is interpreted as giving support to the assumption that these effects may result from a direct action of ethanol. Ethanol toxicity was assessed by exposing females of different ages to a 50% or a 75% (v/v) solution for 60 min and counting the surviving flies 24 h later. The surviving fraction decreased steeply from 1-day-old (100%) to 5-day-old females (1.8%). It is suggested that toxicity may have been due to the action of a metabolite of ethanol, probably acetaldehyde.     

Drosophila Ctr1A functions as a copper transporter essential for development

Copper is an essential trace element required by all aerobic organisms as a cofactor for enzymes involved in normal growth, development, and physiology. Ctr1 proteins are members of a highly conserved family of copper importers responsible for copper uptake across the plasma membrane. Mice lacking Ctr1 die during embryogenesis from widespread developmental defects, demonstrating the need for adequate copper acquisition in the development of metazoan organisms via as yet uncharacterized mechanisms. Whereas the fruit fly, Drosophila melanogaster, expresses three Ctr1 genes, ctr1A, ctr1B, and ctr1C, little is known about their protein isoform-specific roles. Previous studies demonstrated that Ctr1B localizes to the plasma membrane and is not essential for development unless flies are severely copper-deficient or are subjected to copper toxicity. Here we demonstrate that Ctr1A also resides on the plasma membrane and is the primary Drosophila copper transporter. Loss of Ctr1A results in copper-remedial developmental arrest at early larval stages. Ctr1A mutants are deficient in the activity of copper-dependent enzymes, including cytochrome c oxidase and tyrosinase. Amidation of Phe-Met-Arg-Phe-amides, a group of cardiomodulatory neuropeptide hormones that are matured via the action of peptidylglycine alpha-hydroxylating monooxygenase, is defective in neuroendocrine cells of Ctr1A mutant larvae. Moreover, both the Phe-Met-Arg-Phe-amide maturation and heart beat rate defects observed in Ctr1A mutant larvae can be partially rescued by exogenous copper. These studies establish clear physiological distinctions between two Drosophila plasma membrane copper transport proteins and demonstrate that copper import by Ctr1A is required to drive neuropeptide maturation during normal growth and development.     

Larval ethanol exposure alters adult circadian free-running locomotor activity rhythm in Drosophila melanogaster

Alcohol consumption causes disruptions in a variety of daily rhythms, including the sleep-wake cycle. Few studies have explored the effect of alcohol exposure only during developmental stages preceding maturation of the adult circadian clock, and none have examined the effects of alcohol on clock function in Drosophila. This study investigates developmental and behavioral correlates between larval ethanol exposure and the adult circadian clock in Drosophila melanogaster, a well-established model for studying circadian rhythms and effects of ethanol exposure. We reared Drosophila larvae on 0%, 10%, or 20% ethanol-supplemented food and assessed effects upon eclosion and the free-running period of the circadian rhythm of locomotor activity. We observed a dose-dependent effect of ethanol on period, with higher doses resulting in shorter periods. We also identified the third larval instar stage as a critical time for the developmental effects of 10% ethanol on circadian period. These results demonstrate that developmental ethanol exposure causes sustainable shortening of the adult free-running period in Drosophila melanogaster, even after adult exposure to ethanol is terminated, and suggests that the third instar is a sensitive time for this effect.     

Effect of the wingless (wg1) mutation on wing and haltere development in Drosophila melanogaster

Genetic and developmental studies of wingless (wg¹), a new second chromosome recessive mutation in Drosophila melanogaster, have shown that it affects not only wing and haltere development (giving rise to wingless and/or halterless flies), but also results in various abnormalities of the mesothorax. The larvae destined to develop into wingless and/or halterless flies possessed underdeveloped mesothoracic and/or metathoracic imaginal discs.     

Correct developmental expression of a cloned alcohol dehydrogenase gene transduced into the Drosophila germ line

We have used P-element-mediated transformation to introduce a cloned Drosophila alcohol dehydrogenase (Adh) gene into the germ line of ADH null flies. Six independent transformants expressing ADH were identified by their acquired resistance to ethanol. Each transformant carries a single copy of the cloned Adh gene in a different chromosomal location. Four of the six transformant lines exhibit normal Adh expression by the following criteria: quantitative levels of ADH enzyme activity in larvae and adults; qualitative tissue specificity; the size of stable Adh mRNA; and the characteristic developmental switch in utilization of two different Adh promoters. The remaining two transformants express ADH enzyme activity with the correct tissue specificity, but at a lower level than wild type. These results demonstrate that an 11.8 kb chromosomal fragment containing the Adh gene includes the cis-acting sequences necessary for its correct developmental expression, and that a variety of chromosomal sites permit proper Adh gene function.     

Acetaldehyde detoxification mechanisms in Drosophila melanogaster adults involving aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) enzymes

The mechanism of acetaldehyde detoxification in Drosophila melanogaster adults has been studied by comparing physiological in vitro and in vivo data. ADH⁺ and ADH⁻ flies, both lacking aldehyde dehydrogenase activity from ADH (ALDH^ADH, ALDH (ALDH) or both enzymes were exposed to acetaldehyde or ethanol, and the toxicity and internal accumulation of both compounds were determined. Acetaldehyde was extremely lethal for flies whose ALDH activity had been inhibited by cyanamide, though acetaldehyde was effectively detoxified by flies whose ALDH^ADH activity had been inhibited by acetone. After exposure to acetaldehyde, both acetaldehyde and ethanol rapidly accumulated in flies lacking ALDH activity, but not in flies lacking ALDH^ADH activity. However, ethanol but not acetaldehyde quickly accumulated in flies lacking ALDH activity after exposure to ethanol. Our results provide in vivo evidence that, as opposed to larvae, in D. melanogaster adults acetaldehyde is mainly oxidized into acetate by means of ALDH enzymes. However, the reducing activity of the ADH enzyme, which transforms acetaldehyde into ethanol, also plays an essential role in the detoxification of acetaldehyde. Differences in ALDH activity might be important to explain the differences in ethanol tolerance found in natural populations.     

The structure of the normal puparium and in segmentation disorders in Drosophila

In Drosophila puparium is a cuticular mould of external structures formed by the larval cells and can, therefore, preserve traces of developmental defects which appeared in embryogenesis. In this way ontogenetic relationship between embryonic defects and developmental defects in adult individuals can be established. The normal pattern of segmentation in puparia of Drosophila melanogaster was established by comparing segmentation defects in adult flies and corresponding puparia. Boundaries of segment and compartments have been determined, imaginal rudiments of abdominal segments (nests of histoblasts) and rudiments of trachea have been localized on puparia.     

KNOCKDOWN OF ATPsyn‐b CAUSED LARVAL GROWTH DEFECT AND MALE INFERTILITY IN Drosophila

The ATPsyn‐b encoding for subunit b of ATP synthase in Drosophila melanogaster is proposed to act in ATP synthesis and phagocytosis, and has been identified as one of the sperm proteins in both Drosophila and mammals. At present, its details of functions in animal growth and spermatogenesis have not been reported. In this study, we knocked down ATPsyn‐b using Drosophila lines expressing inducible hairpin RNAi constructs and Gal4 drivers. Ubiquitous knockdown of ATPsyn‐b resulted in growth defects in larval stage as the larvae did not grow bigger than the size of normal second‐instar larvae. Knockdown in testes did not interrupt the developmental excursion to viable adult flies, however, these male adults were sterile. Analyses of testes revealed disrupted nuclear bundles during spermatogenesis and abnormal shaping in spermatid elongation. There were no mature sperm in the seminal vesicle of ATPsyn‐b knockdown male testes. These findings suggest us that ATPsyn‐b acts in growth and male fertility of Drosophila.     

Modulation by ellagic acid of DCA-induced developmental toxicity in the zebrafish (Danio rerio)

The ability of ellagic acid (EA) to modulate dichloroacetic acid (DCA)-induced developmental toxicity and oxidative damage was examined in zebrafish embryos. Embryos were exposed to 20 mM EA administered concomitantly with 32 mM DCA at 4 hours postfertilization (hpf) and 20 h later. Embryos were observed through 144 hpf for developmental malformations, and production of superoxide anion (SA) and nitric oxide (NO) was determined in embryonic homogenates. DCA was shown to produce developmental abnormalities and significant levels of SA and NO in zebrafish embryos. EA exposure alleviated the developmental malformations observed in treated embryos and decreased the levels of SA and NO in those same embryos. Less than 10% of DCA + EA exposed embryos showed developmental malformations compared to 100% of embryos treated with DCA alone. Animals in this group that developed malformations were shown to have fewer defects than those treated with DCA only. Taken together, the results confirm the involvement of oxidative stress in the developmental toxicity of DCA in zebrafish embryos, and suggest possible protection against those effects with the use of antioxidants.     

Natural hyperthermia and expression of the heat shock protein Hsp70 affect developmental abnormalities in Drosophila melanogaster

We demonstrate that natural heat stress on wild larval Drosophila melanogaster results in severe developmental defects in >10% of eclosing adults, and that increased copy number of the gene encoding the major inducible heat shock protein of D. melanogaster, Hsp70, is sufficient to reduce the incidence of such abnormalities. Specifically, non-adult D. melanogaster inhabiting necrotic fruit experienced severe, often lethal heat stress in natural settings. Adult flies eclosing from wild larvae that had survived natural heat stress exhibited severe developmental anomalies of wing and abdominal morphology, which should dramatically affect fitness. The frequency of developmental abnormalities varied along two independent natural thermal gradients, exceeding 10% in adults eclosing from larvae developing in warm, sunlit fruit. When exposed to natural heat stress, D. melanogaster larvae with the wild-type number of hsp70 genes (n=10) developed abnormal wings significantly more frequently than a transgenic sister strain with 22 copies of the hsp70 gene. Received: 19 April 1999 / Accepted: 16 July 1999     

The Batten disease Palmitoyl Protein Thioesterase 1 gene regulates neural specification and axon connectivity during Drosophila embryonic development

Palmitoyl Protein Thioesterase 1 (PPT1) is an essential lysosomal protein in the mammalian nervous system whereby defects result in a fatal pediatric disease called Infantile Neuronal Ceroids Lipofuscinosis (INCL). Flies bearing mutations in the Drosophila ortholog Ppt1 exhibit phenotypes similar to the human disease: accumulation of autofluorescence deposits and shortened adult lifespan. Since INCL patients die as young children, early developmental neural defects due to the loss of PPT1 are postulated but have yet to be elucidated. Here we show that Drosophila Ppt1 is required during embryonic neural development. Ppt1 embryos display numerous neural defects ranging from abnormal cell fate specification in a number of identified precursor lineages in the CNS, missing and disorganized neurons, faulty motoneuronal axon trajectory, and discontinuous, misaligned, and incorrect midline crossings of the longitudinal axon bundles of the ventral nerve cord. Defects in the PNS include a decreased number of sensory neurons, disorganized chordotonal neural clusters, and abnormally shaped neurons with aberrant dendritic projections. These results indicate that Ppt1 is essential for proper neuronal cell fates and organization; and to establish the local environment for proper axon guidance and fasciculation. Ppt1 function is well conserved from humans to flies; thus the INCL pathologies may be due, in part, to the accumulation of various embryonic neural defects similar to that of Drosophila. These findings may be relevant for understanding the developmental origin of neural deficiencies in INCL.     

Reduction of endoplasmic reticulum stress attenuates the defects caused by Drosophila mitofusin depletion

Ablation of the mitochondrial fusion and endoplasmic reticulum (ER)–tethering protein Mfn2 causes ER stress, but whether this is just an epiphenomenon of mitochondrial dysfunction or a contributor to the phenotypes in mitofusin (Mfn)-depleted Drosophila melanogaster is unclear. In this paper, we show that reduction of ER dysfunction ameliorates the functional and developmental defects of flies lacking the single Mfn mitochondrial assembly regulatory factor (Marf). Ubiquitous or neuron- and muscle-specific Marf ablation was lethal, altering mitochondrial and ER morphology and triggering ER stress that was conversely absent in flies lacking the fusion protein optic atrophy 1. Expression of Mfn2 and ER stress reduction in flies lacking Marf corrected ER shape, attenuating the developmental and motor defects. Thus, ER stress is a targetable pathogenetic component of the phenotypes caused by Drosophila Mfn ablation.     

Lethality and Developmental Delay in Drosophila melanogaster Larvae after Ingestion of Selected Pseudomonas fluorescens Strains

Background

The fruit fly, Drosophila melanogaster, is a well-established model organism for probing the molecular and cellular basis of physiological and immune system responses of adults or late stage larvae to bacterial challenge. However, very little is known about the consequences of bacterial infections that occur in earlier stages of development. We have infected mid-second instar larvae with strains of Pseudomonas fluorescens to determine how infection alters the ability of larvae to survive and complete development.

Methodology/Principal Findings

We mimicked natural routes of infection using a non-invasive feeding procedure to study the toxicity of the three sequenced P. fluorescens strains (Pf0-1, SBW25, and Pf-5) to Drosophila melanogaster. Larvae fed with the three strains of P. fluorescens showed distinct differences in developmental trajectory and survival. Treatment with SBW25 caused a subset of insects to die concomitant with a systemic melanization reaction at larval, pupal or adult stages. Larvae fed with Pf-5 died in a dose-dependent manner with adult survivors showing eye and wing morphological defects. In addition, larvae in the Pf-5 treatment groups showed a dose-dependent delay in the onset of metamorphosis relative to control-, Pf0-1-, and SBW25-treated larvae. A functional gacA gene is required for the toxic properties of wild-type Pf-5 bacteria.

Conclusions/Significance

These experiments are the first to demonstrate that ingestion of P. fluorescens bacteria by D. melanogaster larvae causes both lethal and non-lethal phenotypes, including delay in the onset of metamorphosis and morphological defects in surviving adult flies, which can be decoupled.     

Effects of protein deficiency on the teratogenicity of cytochalasins in mice

The developmental toxicity of cytochalasins B (CB) and D (CD) was evaluated in protein-deprived mice. Pregnant CD-1 mice were assigned to control (26%), 16%, 8%, or 4% dietary protein groups on gestation day 1 and dosed by gavage with 0 or 1.5 mg/kg CB or CD on gestation day 8 (plug = day 1). They were killed and subjected to teratological examination on day 18. CD, but not CB, increased prenatal mortality but failed to interact significantly with dietary protein level. Fetal weights were decreased in the 4% and 8% dietary protein groups, but cytochalasin treatment did not exacerbate this effect. Cytochalasin treatment was associated with gross fetal malformations, primarily neural tube defects. Although CB and CD did not significantly increase the percentage of grossly malformed fetuses per litter, the data was suggestive of such an effect, and the incidence of affected litters was increased by cytochalasin treatment in all but the 4% protein group. Skeletal defects, such as jaw malformations, rib or sternebrae variations, and unossified skull bones appeared to be increased by both cytochalasin treatment and dietary protein deficiency. The differences from control values were nonsignificant, however, except for some cases of cytochalasin effects on skull ossification. These results show a general lack of effect of protein deprivation on the developmental toxicity of cytochalasins.     

Developmental changes in Drosophila melanogaster following exposure to alternating electromagnetic fields

This study investigated the biological effects of alternating electromagnetic fields (EMFs) on developmental stages of Drosophila melanogaster eggs and the first, second and third instar larvae stages. D. melanogaster eggs and larval stages were exposed to a 11 mT 50 Hz field produced by a pair of Helmholtz coils. Each stage was exposed to aEMFs for 2, 4, 6 and 8 h. Features of adult flies such as head, thorax, abdomen and other morphological changes were studied and compared. The frequency of abnormal flies was calculated using statistical methods at P <.05. The results obtained from exposing larvae in different stages of development showed a significant increase in the number of abnormal adult flies, whereas no significant increase was observed in the group arising from eggs exposed to aEMFs. Also, it appeared that duration of exposure correlates with the increase in the number of abnormal flies. There was no significant difference in mortality rate and sex distribution of the abnormal flies between field exposed and the control groups.