Xenobiotic-metabolizing enzymes and benzo[a]pyrene metabolism in the benzo[a]pyrene-sensitive mutant strain of Drosophila simulans.

Basal levels of aryl hydrocarbon hydroxylase, epoxide hydrolase and glutathione S-transferase enzyme activities, cytochrome P-450 content and inducibility of enzymes with phenobarbital were found to be similar in the microsomes of D. simulans mutant strain 364yv, which is sensitive to the toxic and mutagenic effects of benzo[a]pyrene (BP), and of the wild resistant Turku strain. In contrast, increases in the rate of BP turnover per molecule of cytochrome P-450, intensity of the hemoprotein band with apparent molecular weight 56,000 and the yield of BP 7,8-dihydrodiol and 9,10-dihydrodiol occurred only in microsomes of BP-pretreated 364yv flies but not of Turku ones. It is likely that BP induces an aberrant form of cytochrome P-450 in 364yv flies with a rare mutation in one of the P-450 regulating genes.     

Benzo(a)pyrene pretreatment of Drosophila simulans mutant strain results in the induction of aberrant isoform of cytochrome P-450 with increased capacity to metabolize benzo(a)pyrene]

The basal level of benzo(a)pyrene monooxygenase, epoxide hydrolase and glutathione S-transferase activity as well as the content of cytochrome P-450 were found the same in both compared benzo(a)pyrene (BP) sensitive D. simulans strain 364yv and BP-resistant wild one (Turku). Phenobarbital pretreatment resulted in the same increase level of these enzyme activities in both strains. BP-pretreatment of 364yv flies decreased the amount of the cytochrome P-450 but raised up the turnover of BP per molecule of cytochrome P-450. SDS-polyacrylamide gel electrophoresis of the microsomal proteins from BP-pretreated 364yv flies (but not from Turku) showed an increased hemoprotein content in the 56000 band. The relationship between BP-sensitivity of the strain 364yv and BP-induced aberrant isoform of the cytochrome P-450 has been discussed.     

Chromosome mapping of Rmp-4, a gonad-dependent gene encoding host resistance to mousepox.

DBA/2 (D2) mice are susceptible and C57BL/6 (B6) mice are resistant to lethal mousepox. A congenic resistant strain, D2.B6-Rmp-4r (D2.R4), was developed by serially backcrossing male mice that survived ectromelia virus infection with D2 mice, beginning with (B6 x D2)F1 mice. Male D2.R4 mice were at least 300-fold more resistant to lethal mousepox than male D2 mice. Female D2.R4 mice were 100-fold more resistant than male D2.R4 mice and 500-fold more resistant than female D2 mice. Neonatal gonadectomy prevented development of resistance in D2.R4 mice of both sexes. Differences in resistance between strains and between sexes correlated with restriction of virus replication in spleen and liver, but gender differences were less evident in liver than in spleen. High-resolution interval mapping of the 19 autosomes of D2.R4 mice using dispersed informative microsatellites as marker loci revealed a segment of distal chromosome 1 to be of B6 origin. Haplotypes for a marker locus, D1Mit57, from the differential segment were determined in (D2.R4 x D2)F1 x D2 backcross mice, which were then infected with ectromelia virus. Significantly more heterozygotes than homozygotes survived ectromelia virus infection in both sexes. Whereas nearly all surviving males were heterozygotes, 44% of surviving females were homozygotes. These results indicate that resistance in D2.R4 mice is determined by a gonad-dependent gene on distal chromosome 1, provisionally named Rmp-4, and by an ovary-dependent factor that is not genetically linked to Rmp-4.     

Induction of tumor clones in D. melanogaster wts/+ heterozygotes with chemical carcinogens.

Ten chemicals were assessed for blastomogenic activity in adult wts/+ heterozygotes of D. melanogaster. All of the strong mammalian carcinogens tested (benzo(a)pyrene (B(a)P), pyrene, aflatoxin B(1), 2-acetylaminofluorene (2-AAF) and cis-dichlorodihydroxydiamminoplatinum IV) were also shown to be strong Drosophila blastomogens. They induced several times more tumors than their counterparts that are less carcinogenic for mammals (4-acetylaminofluorene (4-AAF), aflatoxins B(2) and G(2)) and 4-(methylnitrosamino)-1-(-3-pyridine)-1-butanone (NNK). Benzo(e)pyrene (B(e)P) and pyrene demonstrated minor effects. Most tumors were localized on the wing and notum, which are the derivatives of the wing disc. Humeri derived from dorsal prothoracic disc and the abdominal tergites and sternites had the lowest number of tumors. The tumor frequency in the cross of the wild type females with wts(P2)/TM6B males was different from that in the reciprocal cross. The former type of cross exhibited consistently higher tumor frequency both in the experimental and control series.     

Genotoxic potency in Drosophila melanogaster of selected aromatic amines and polycyclic aromatic hydrocarbons as assayed in the DNA repair test

Drosophila melanogaster stock consisting of meiotic recombination deficient (Rec⁻) double mutant mei-9^a mei-41^D5 males and Rec⁺ females was exposed at the larval stage to an aromatic amine or a polycyclic aromatic hydrocarbon. After emergence as adult flies, the males and the females were scored separately. When the treatment caused a dose-dependent reduction in the male to female ratio from the control level, the experiment was repeated with a larval stock consisting of Rec⁺ males and Rec⁺ females under comparable conditions. A preferential killing effect upon Rec⁻ larvae was taken as evidence of DNA damaging effect of the test compound. Among 16 compounds tested, 1-AP, B(a)P, 2-AF, DAF, 4-AAF, 2-AAF, 1-AA, 2-AA, DMA, B(a)A and DMBA were registered as positive; Py and 3-MC were weakly positive; and B(e)P, Fluo and Ant were negative. The selective killing effects of the compounds in each of the pyrene, fluorene and anthracene series varied drastically as a function of structure in a way similar to that reported for the genotoxicity in Drosophila and the carcinogenicity in rodents. The Drosophila DNA repair assay will serve as a simple adjunct to the already available means for studying the genotoxic potency of aromatic amines and polycyclic aromatic hydrocarbons.     

The specificity of the genotoxic action of carcinogenic aromatic compounds on Drosophila mus mutants]

Hypersensitivity to the toxic effect of benzo(a)pyrene (B(a)P) was determined in homozygous larvae of two D. melanogaster mus strains (mus 208B2 and mus210). The two others (mus205B1 and mus208B1) were found to be less sensitive and the parent strain was resistant. The lack of correlation between the sensitivity in larvae and the activity of aryl hydrocarbon hydroxylase in S15 fractions from adult flies whole body homogenates of the same strains was demonstrated. The hypertoxic effect of B(a)P and 2-acethylaminofluorene in strain mus210 seems to be rather specific because noncarcinogenic pyrene, benzo(e)pyrene and fluorene did not affect the survival of this most sensitive strain. Perspectives of the strain mus210 use for the environmental genotoxic pollutants screening were discussed.     

Female mice of DDK strain are fully fertile in the intersubspecific crosses with Mus musculus molossinus and M. m. castaneus

The female mice of DDK strain are almost infertile when mated with males from other strains. This phenomenon is caused by the early death of F1 embryos owing to the incompatibility system attributed to the ovum mutant (Om) locus on Chromosome (Chr) 11 and known as DDK syndrome. In the present study, DDK females were found to be fully fertile in the intersubspecific matings with the males of two wild mouse-derived strains, MOM (originated from Japanese wild mice, Mus musculus molossinus) and Cas (originated from Philippine wild mice, M. m. castaneus), indicating that no incompatibility exists between DDK oocytes and spermatozoa of MOM and Cas strains. Furthermore, this compatibility has been confirmed by the following two findings: (1) Normal fertility was shown by the two types of backcrosses, DDK females x F(1) (DDK female x MOM male) males and DDK females x F(1) (DDK female x Cas male) males; and (2) the offspring from these backcrosses segregated equally into the homozygotes and heterozygotes as genotyped by the microsatellite markers closely linked to Om locus. MOM and Cas strains would be useful for further investigations on the Om locus. On the other hand, the litter size of F(1) [C57BL/6Cr (B6) female x Cas male] females mated with B6 males was about half that of the mating with DDK males. It would be interesting to investigate whether this reduction in fertility is related to the Om locus or not.     

Mutation of Indy(p115) increases the life expectancy of imago of Drosophila melanogaster depending on sex and genetic background

The Indyp115 allele in heterozygous state almost doubles the life span of adult Drosophila melanogaster, and this effect largely depends on the strain used for obtaining heterozygotes. Male and female life span depends on Indyp115 to a different degree. Apart from Indyp115 heterozygotes, sexual dimorphism for life span was also observed in strain Hikone-AW, but not in OregonR and TM3 balancer. In heterozygotes Indyp115/OR, both the average life span and female reproductive period increased as compared to OR control. No substantial increase in female reproductive period in Indyp115/Hk heterozygotes was found. In homozygotes for allele Indyp115, we have previously revealed two phases of embryotic lethality and lethality at the larval and pupa stages. Thus, allele Indyp115 has a double and opposite effect on Drosophila viability. On the one hand, it extends the life span of adult flies, on the other, decreases survival at preimaginal stages.     

Genetic basis for resistance to gel baits, fipronil, and sugar-based attractants in German cockroaches (Dictyoptera: blattellidae)

A gel bait-resistant Blattella germanica (L.) strain (Cincy) was collected in Cincinnati, OH, in 2003. This strain exhibited strong behavioral resistance to Avert (0.05% abamectin), Maxforce FC (0.01% fipronil), and Pre-Empt (2.15% imidacloprid) gel baits. Reciprocal mass crosses and back crosses between the Cincy strain and a susceptible strain (Jwax) were made and tested for their inheritance of resistance to Avert, Maxforce FC, and Pre-Empt gel baits. Topical assays comparing the parental and reciprocal-heterozygous strains indicated the resistance to fipronil was incompletely recessive. LD50 and LD50 values of the Jwax male x Cincy female strain were not significantly different from he Jwax female x Cincy male strain, suggesting no sex linkage in physiological fipronil resistance. Feeding assays revealed that F1 reciprocal crosses were significantly less responsive to blank Avert and Maxforce FC baits (without active ingredients) than the susceptible strain. The Jwax female x Cincy male strain did not display significantly greater consumption of blank Avert and Maxforce FC baits relative to the Jwax male x Cincy female strain. In feeding assays with agar containing D-fructose, D-galactose, D-glucose, D-lactose, D-maltose, and D-sucrose, the crosses showed an intermediate feeding response to glucose compared with the Cincy and Jwax strains, and a similar response to other sugars compared with the Jwax strain. The Jwax male x Cincy female strain was significantly less responsive to glucose than the Jwax female x Cincy male strain. Mortality induced by Avert, Maxforce FC, and Pre-Empt gel baits against the F6 Jwax male x Cincy femaale strain was 44.2 +/- 6.8, 92.9 +/- 2.1, and 78.7 +/- 5.2%, respectively, indicating the resistance to Avert and Pre-Empt gel baits inherited by Cincy females was extremely stable. The F6 Jwax male x Cincy female strain was significantly more resistant to Avert, Maxforce FC, and Pre-Empt than the F6 Jwax female x Cincy male strain. These findings suggest that behavioral resistance to gel baits has weak sex-linkage, with a greater degree of the resistance trait being inherited by female cockroaches. Alternatively, physiological resistance to fipronil has no sex-linkage, but it is nonetheless important to the complete resistance phenotype.     

Mutagenicity of amine drugs and their products of nitrosation

Mouse spot tests using dimethylbenz[a]anthracene (DMBA) were carried out on PW strain male mice and female C57BL/6 mice. DMBA induced somatic gene mutations in developing mouse embryonic cells. Pretreatment of pregnant females with phenobarbital (PB) reduced the incidence of somatic mutation by DMBA. The testes of males treated with DMBA in utero, whether treated with PB or not, showed severe retardation of development. The amount of cytochrome p-450 in the liver of C57BL/6 females increased about 2-fold by the pretreatment schedule, carried out on days 10-12 of pregnancy.     

Effect of synergists on the oral and topical toxicity of azamethiphos to organophosphate-resistant houseflies (Diptera: Muscidae).

Dermal and oral toxicities of azamethiphos were determined in two organophosphate-resistant and one susceptible strain of houseflies, Musca domestica L. The 594vb strain was 1,967-fold more resistant to azamethiphos when compared with the susceptible Chemical Specialties Manufacturers Association (CSMA) strain by dermal application. When the compound was administered orally to the 594vb strain, we observed only a 15-fold resistance. In contrast, the Yachiyo strain, which show 1,500-fold resistance to diazinon and which has known multiple mechanisms for organophosphate resistance, showed only 6-fold resistance to azamethiphos by topical application and 4-fold resistance by oral administration. Azamethiphos administered dermally and orally was equally toxic to the CSMA and Yachiyo strains. However, when azamethiphos was administered to the 594vb strain, the insecticide was 71 times more toxic orally than by the dermal route. This result indicated involvement of a cuticular penetration factor in the resistance mechanism. The effect on azamethiphos toxicity of piperonyl butoxide (PB), an inhibitor of the monooxygenases, and tributylphosphorotrithioate (DEF), an esterase inhibitor, was investigated in the three strains. Pretreatment of the flies with PB, DEF, or PB+DEF before topical application of azamethiphos resulted in a significant decrease in LD50s in all the strains. The degree of synergism, however, varied depending upon the strains and the synergists. Similar results were obtained when azamethiphos was administered orally following pretreatment of the flies with PB+DEF. We attribute the high level of azamethiphos resistance in the 594vb strain partly to increased degradation by oxidative and hydrolytic enzymes. The hydrolytic enzymes are more important, but other factors including reduced cuticular penetration and insensitive acetylcholinesterase may be involved.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of mycotoxin pretreatment on aflatoxin B1 post-treatment toxicity in Drosophila melanogaster (Diptera)

Two wild-type laboratory strains of Drosophila melanogaster were used in this study: strain Flordia-9, which is sensitive to aflatoxin B₁ (AFB₁)-induced toxicity, and strain Lausanne-S, which is resistant. Eggs of these strains were deposited on medium containing either low or high doses of dietary AFG₁, AFB₂, or sterigmatocystin (ST) and allowed to develop into second instar larvae. After this pretreatment, the larvae were transferred onto medium containing either high or low doses of dietary AFB₁ (post-treatment) and allowed to complete development and eclose as adults. Viability and development data were analyzed to determine the effects of the various pretreatments on the level of AFB₁-induced toxicity in the post-treatments. In no case did any of the pretreatments reduce the toxic effects of AFB₁ post-treatment responses. However, for strain Florida-9, all high-dose pretreatments resulted in enhanced post-treatment toxicity, and all low-dose pretreatments also enhanced toxicity of high-dose post-treatments. For strain Lausanne-S, high-dose AFB₂ pretreatment significantly enhanced toxicity of both high- and low-dose post-treatments. These results indicate that, in strain Florida-9, pretreatment with relatively less toxic mycotoxins (ST and AFB₂) has an enhancing effect on AFB₁-induced toxicity, whereas in strain Lausanne-S, a similar but smaller enhancing effect is seen only with AFB₂ pretratment.     

Sensitivity differences displayed by Drosophila melanogaster larvae of different ages to the toxic effects of growth on media containing aflatoxin B1.

Using Drosophila melanogaster, the relative sensitivities of various larval stages to the toxic effects of growth on media supplemented with either 0.44 or 0.88 ppm aflatoxin B1 (AFB1) were determined. Two strains of fruit flies were tested: strain A-11 which is relatively resistant to AFB1 induced toxicity, and strain A-9 which is quite sensitive. Eggs, mid-first, mid-second and early-, mid- and late-third instar larvae were transferred onto AFB1 media and allowed to complete larval and pupal development and eclose as adults. At the 0.44 ppm concentration, strain A-11 showed no effect, while only first instar larvae of strain A-9 showed significant mortality rates for first instar larvae, but the A-9 larvae die at higher rates than the A-11 larvae. In addition, second and third instar larvae of strain A-9 show significant mortality rates when grown at 0.88 ppm AFB1, while these stages are not affected in strain A-11.     

Genetic sexing in Drosophila melanogaster using the alcohol dehydrogenase locus and a Y-linked translocation

Summary By incorporating ethanol (4% v/v) into the larval rearing medium of a specially constructed Drosophila melanogaster strain it was possible to produce only male adults; the female larvae died.In this strain, the male determining chromosome was linked with a positive Alcohol dehydrogenase (ADH) allele by a translocation. The females were homozygous for the null allele and hence sensitive to ethanol.This genetic sexing method is discussed in relation to its use in the genetic control of insects.     

Discriminating activation of CYP2B9 expression in male C57BL/6 mouse liver by beta-estradiol

The inducible expression of the cytochrome P450 2B subfamily was investigated in male C57BL/6 (B6) and DBA/2 (D2) mice, as well as their hybrids, B6D2F1, at the mRNA level. The expression of hepatic CYP2B mRNAs in B6 was lightly induced by beta-estradiol (ES), while that by phenobarbital (PB) or 1,1,1-trichloro-2, 2-bis(p-chlorophenyl) ethane (DDT) was prominent. Discriminating analysis showed a novelty that ES markedly induced CYP2B9 mRNA expression, whereas PB and DDT increased CYP2B10 more than CYP2B9 expression: albeit both mRNA species responded to all three inducers. Furthermore, the specific induction by ES of CYP2B9 mRNA in B6 male mice, but not D2 male mice, suggests strain dependency in the regulatory pathway of CYP2B9 expression.     

The temperature sensitivity of mutations in species of the virilis group of Drosophila. II. The effect of temperature on the maternal effect on the puffed marker following hybridization

The influence of temperature (17 and 31 degrees) on the maternal effect of mutation Puffed (Pu) in Drosophila hybrids has been studied. In the hybrids female D. littoralis +/+ x male D. virilis Pu/Pu, the stage of formation of black ring on anterior spiracles in the 3rd larval instar is sensitive to 31 degrees. In the hybrids female D. virilis Pu/Pu x male D. littoralis +/+, the expression of Pu gene manifests the maternal effect and as a result, two temperature-sensitive stages are found. The first one--onset of embryogenesis (2 to 4 hrs). At the temperature 17 degrees, the penetrance of Pu increases. The second stage is sensitive to 31 degrees and coincides with the period of black rings formation on anterior spiracles in the 3rd laval instar. It has been shown that at least two genetic systems take part in the formation of this feature. One group of genes controls the maternal effect and is sensitive to low temperature in the early embryogenesis of the hybrids female D. virilis x male D. littoralis. The second one--Pu gene and its modifiers--is active during the 2nd half of the 3rd larval instar and is heat-sensitive.     

Strain-differences and inducibility of microsomal oxidative enzymes in Drosophila melanogaster flies

Some basic characteristics of the enzyme system involved in the oxidative metabolism of xenobiotic compounds were investigated in Drosophila melanogaster flies. Attention was focussed on (1) the normal levels of these enzymes and their activities in whole flies, in different parts of the fly's body and in different sexes, (2) the changes in levels and activities of the enzymes elicited by pretreatment of the flies with known enzyme inducers and (3) differences between strains.Four commonly used wild-type (WT) strains, three insecticide resistant strains (IR) and one white-eyed mutant strain were employed. Except in those experiments on sex differences and in spatial distribution in the fly's body of the enzymatic activities, microsomes were isolated from whole-body homogenates of mixtures of female and male flies. Microsomal cytochrome P-450, benzo[a]pyrene (BP) hydroxylation, p-nitroanisole (pNA) demethylation and aminopyrine (AP) demethylation were measured in control flies and in flies pretreated with Aroclor 1254 (AC), phenobarbital (PB) or butylated hydroxytoluene (BHT).In flies of the WT strain Berlin-K, there were no significant differences in BP hydroxylation activity and its inducibility between the two sexes. In males, inducibility of BP hydroxylation activity was similar in the head, thorax and abdomen, but significantly lower in testis. Considerable differences in some enzyme activities were found between the strains. pNA demethylation and AP demethylation were substantially higher in all IR strains, while no correlation could be found between their increased insecticide resistance and BP hydroxylating capacity or cytochrome P-450 content of the microsomes.Response to enzyme inducing compounds was found to be strain-dependent. PB proved to be a more efficient inducer of BP hydroxylation than AC, which does induce pNA demethylation. BHT has inducing properties that are intermediate between PB and AC. IR strain Hikone-R turned out to be an exception, possessing very low BP hydroxylating capacity and a low degree of inducibility of mixed-function oxidase activities. Differential temperature dependence was found for BP hydroxylation as compared with pNA demethylation. While BP hydroxylation was doubled when raising the temperature from 25°C to 35°C, pNA demethylation was reduced by 50%.     

Antipyrine metabolism in the rat by three hepatic monooxygenases

The relative contribution of 3 oxidative reactions of antipyrine to its metabolism in vivo were assessed by comparing male and female rats, and by studying the effects of phenobarbital (PB) and 3-methylcholanthrene (MC). After [N¹⁴CH₃] antipyrine, 11–15% of the dose was excreted as ¹⁴CO₂ in both sexes as a consequence of N-demethylation. PB pretreatment had no effect but MC doubled ¹⁴CO₂ excretion. The other metabolites, 4-hydroxyantipyrine (4OHA) and 3-hydroxymethylantipyrine (3HMA), in urine were determined by thin-layer chromatography. Of the total activity in the urine, 4OHA represented 30% in male and 40% in female rats; 3HMA represented 35% in male and 20% in female rats. The ratio of 4OHA to 3HMA in both sexes increased after PB and MC, the effect being more pronounced with the latter. The results show that the 3 major oxidative pathways of antipyrine metabolism are mediated by different enzymes, almost certainly different forms of cytochrome P450.     

Chromosomal effects on male and female components of sperm precedence in Drosophila

Recent experiments with Drosophila have demonstrated that the success of sperm in multiply mated females depends on the genotype of both the male and the female. To further characterize the distinction between male and female roles in sperm success, we scored variation in both sexes in sperm competitive ability among a set of chromosome replacement lines that allow identification of effects to each chromosome. We detected significant male and female effects on sperm precedence, defined as the ability of a male's ejaculate to displace resident sperm (P2) or avoid being displaced by subsequent matings (P1). Tests of effects of first, second and third chromosome substitutions revealed significant differences among third chromosomes in male sperm precedence (both P1 and P2) and a first x second chromosome interaction in female's effect on sperm precedence (only P1). We found no significant correlation between male and female effects on sperm precedence, suggesting that the variation found in both P1 and P2 has a different genetic cause in the two sexes.     

Field resistance of codling moth against Cydia pomonella granulovirus (CpGV) is autosomal and incompletely dominant inherited

The current appearance of local codling moth populations with resistance to Cydia pomonella granulovirus (CpGV) is an impediment to continuous CpGV application. Therefore, crossing experiments have been performed in order to gain information about the inheritance of resistance. Evidence is presented that the observed field resistance is stably inherited even under non-selective conditions in the laboratory. Offspring of reciprocal F(1) crosses between a susceptible ('S') and a resistant ('R') strain and backcrosses between F(1) and S were bioassayed at different CpGV concentrations. The resistant strain showed 100 times lower susceptibility in 7-day bioassays. The responses of the reciprocal crosses (male S x female R and female S x male R) did not differ significantly, indicating that resistance is autosomally inherited. The median lethal concentration for the F(1) progeny was intermediate between those of its parental strains. Mortality data obtained from the backcrosses suggested that inheritance of resistance is due to a non-additive, polygenic trait.