Genotoxic activation of hydrazine, two dialkylhydrazines, thiourea and ethylene thiourea in the somatic w/w+ assay of Drosophila melanogaster

Genotoxic activation of hydrazine (HZ), two symmetrical dialkylhydrazines, namely, 1,2dimethylhydrazine and 1,2-diethylhydrazine (SDMH and SDEH), thiourea (TU) and ethylene thiourea (ETU) has been evaluated by means of the w/w+ somatic assay of Drosophila. Both low bioactivation insecticide-susceptible (IS) and high biotransformation insecticide-resistant (IR) strains were used. The combined application of insecticide-susceptible and insecticide-resistant strains should, in principle, detect somatic cell recombinagens in the Drosophila melanogaster in vivo w/w+ assay. The IS strain was more susceptible to toxicity induced by the test chemicals than the IR stocks. Its performance in the biotransformation of the chemicals tested was rather poor. TU was inactive in all strains. With the active compounds, spot frequencies increased approximately linearly with dose for each spot type. SDEH gave a strong positive result in all three female genotypes exposed. HZ, ETU and SDMH were overall weakly positive in the IR strain Haag-79 (HG-R). Interestingly, ETU was clearly positive in the IR Hikone-R (HK-R) strain. A comparison of the recombinagenic potencies between the active and the weakly positive compounds, and among strains, showed pronounced genotype-dependent differences between the low and the high bioactivation strains. The ability of Drosophila to express several procarcinogens in relation to insecticide-resistance after activation catalyzed by CYP450 enzymes is discussed.     

Mutagenicity of 7,12-dimethyl[a]anthracene and some other aromatic mutagens in Drosophila melanogaster

The optimal conditions for mutagenesis studies with DMBA and some other aromatic carcinogens in Drosophila were investigated in detail. The results presented in this paper indicate the following.The mutagenic effectiveness of DMBA is dependent on the route of administration, injection being far more effective when compared with feeding.The choice of the solvent is a crucial experimental condition. DMBA, when dissolved in oil/DMF, is ineffective whereas a special fat emulsion of DMBA gives high mutation frequencies.There appears to be an extreme strain dependence in the mutagenicity of DMBA. Mutagenic effectiveness in strain Berlin-K was rather low, whereas Oregon-K and Karsnäs-60 proved to be very susceptible to DMBA.Under the conditions of test, DMBA did not induce loss of a ring-X chromosome and did not produce recessive lethal mutations in such a chromosome.DMBA did not produce 2–3 translocations to any significant extent.An increase in DMBA-induced recessive lethal mutations was found upon storage of treated sperm with an optimal storage time of 4–10 days.DMBA is efficient in the production of delayed recessive lethal mutations in strain Berlin-K. Twice as many lethals were recovered with the F3 generation as compared with those in F2. In strain Oregon-K, where the frequency of F2 lethals was much higher than in strain Berlin-K, the ratio of F3/F2 lethals was clearly lower.Enzyme induction with phenobarbital reduces the mutagenic effectiveness of DMBAWith TMBA, similar strain differences in sensitivity were observed as those found for DMBA. Whereas TMBA was not mutagenic in Berlin-K, considerable mutagenicity was observed in Oregon-K and Karsnäs-60.Injection of carcinogenic polycyclic aromatic hydrocarbons and aromatic amines, when dissolved in special fat emulsions, enhances the mutagenic effectiveness of some compounds (DMBA, TMBA, DA and AcO-AAF), but this procedure does not always solve the problems-pertinent to these classes of promutagens in Drosophila.     

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%.     

High expression of Cyp6g1, a cytochrome P450 gene, does not necessarily confer DDT resistance in Drosophila melanogaster

Cytochrome P450 monooxygenases, a family of detoxifying enzymes, are thought to confer resistance to various insecticides including DDT. Daborn et al. [Daborn, P., Yen, J.L., Bogwitz, M., Le Goff, G., Feil, et al. 2002. A single p450 allele associated with insecticide resistance in Drosophila. Science 297, 2253-2256.] suggested that the Accord transposable element causes overexpression of a Cyp6g1 allele, which has spread globally and is the basis of DDT resistance in Drosophila melanogaster populations. To determine whether the same phenomenon also operates in other Drosophila strains, we investigated 91-R, 91-C, ry(506), Wisconsin, Canton-SH and Hikone-RH strains. While the LC(50) values for the 91-R and Wisconsin strains are 8348 microg and 447 microg of DDT, respectively, values for the other four strains range between 0.74 to 20.9 microg. As expected, the susceptible ry(506) and 91-C strains have about 16-33-fold lower levels of CYP6G1 mRNA than the resistant 91-R and Wisconsin strains. Surprisingly, CYP6G1 mRNA and protein levels in the Canton-SH and Hikone-RH strains are as high as in the two resistant strains, yet they are as susceptible as the 91-C strain. The susceptible phenotype of the Canton-SH and Hikone-RH strains is not due to mutation in the Cyp6g1 gene; sequence analysis showed that Cyp6g1 alleles of resistant and susceptible strains are very similar and cannot be classified into resistant and susceptible alleles. As observed by others, we also found that only the 5'-upstream DNA of overexpressing alleles of Cyp6g1 has an insertional DNA, which is similar to Accord and Ninja elements. To examine the role of Cyp6g1 in DDT resistance, we substituted the Cyp6g1 allele of the 91-R strain with the allele from the susceptible 91-C strain via recombination and synthesized three recombinant lines. All three lines lacked Accord insertion and showed low expression of Cyp6g1 like the 91-C strain, yet they were as highly resistant as the 91-R strain. We conclude a strain may not have to have Accord insertion in the Cyp6g1 gene and the Cyp6g1 itself may not have to be overexpressed for DDT resistance to occur.     

Factors on the third chromosome affect the level of cyp6a2 and cyp6a8 expression in Drosophila melanogaster

The expression of two second chromosome-linked cytochrome P450 genes, Cyp6a2 and Cyp6a8, of Drosophila melanogaster was measured in various strains. Six different strains, including ry(506) and 91-C, showed low or undetectable levels of CYP6A2 and CYP6A8 mRNAs, suggesting that low expression is the wild-type phenotype of Cyp6a2 and Cyp6a8 genes. In the 91-R and MHIII-D23 strains, however, both these genes are overexpressed. In order to examine the genetic basis of Cyp6a2 and Cyp6a8 expression, CYP6A2 and CYP6A8 RNA levels were measured in the F1 hybrids of overproducer (91-R and MHIII-D23) and underproducer (ry(506) and 91-C) strains. Results showed that the total amounts of CYP6A2 and CYP6A8 mRNAs in the F1 hybrids were lower than half the amounts of these RNAs found in the overproducer parental strains. This suggested that the underproducer strains carry loci which downregulate Cyp6a2 and Cyp6a8 gene expression. To determine the chromosome linkage of these loci, several stocks homozygous for the second chromosome of overproducer 91-R strain and, therefore, homozygous for the Cyp6a2-91R and Cyp6a8-91R alleles were synthesized. The third chromosomes in all these stocks were from the underproducer ry(506) strain. The levels of expression of both Cyp6a2-91R and Cyp6a8-91R genes in these three stocks were significantly lower than that observed in the 91-R strain. One of these stocks, named iso-2, showing reduced expression, was used to synthesize two new isogenic stocks by resubstituting the third chromosome of ry(506) origin with third chromosomes of the 91-R strain. Expression of both Cyp6a2-91R and Cyp6a8-91R alleles was found to be much higher in these two resubstituted isogenic stocks than in the progenitor iso-2 stock. Taken together, these results suggest that the second chromosome-linked Cyp6a2 and Cypa8 genes are regulated by loci present on the third chromosome, and the wild-type function of these loci is to repress these two Cyp genes. The data also suggest that Cyp6a2 and Cyp6a8 overexpression in the 91-R and MHIII-D23 strains is more likely due to mutation in the repressor locus (or loci) rather than in the cis-regulatory sequences of the Cyp6a2 and Cyp6a8 genes.     

Studies on mutagen-sensitive strains of Drosophila melanogaster. VII. Effects of repair deficiency in males on X-ray-induced sex-linked recessive lethals in spermatozoa

The response of mature spermatozoa to the X-ray induction (500 R and 3000 R) of sex-linked recessive lethals was studied in Drosophila melanogaster males known to be deficient in excision- or post-replication repair of UV damage in somatic cells. The results show that the induced frequencies of recessive lethals in the excision-repair-deficient males (mei-9a and mei-9L1) are similar to those in the appropriate repair-proficient males (mei+ and Berlin-K). However, in the post-replication-repair-deficient males (w mus(1)101D1), these frequencies are significantly lower than in the comparable repair-proficient males (w) after 500 R, but not after 3000 R.     

Female X-chromosome mosaicism for NOX2 deficiency presents unique inflammatory phenotype and improves outcome in polymicrobial sepsis

Cellular X-chromosome mosaicism, which is unique to females, may be advantageous during pathophysiological challenges compared with the single X-chromosome machinery of males, and it may contribute to gender dimorphism in the inflammatory response. We tested the hypothesis of whether cellular mosaicism for the X-linked gp91phox (NOX2) deficiency, the catalytic component of the superoxide anion-generating NADPH oxidase complex, is advantageous during polymicrobial sepsis. Deficient, wild-type (WT), and heterozygous/mosaic mice were compared following polymicrobial sepsis initiated by cecal ligation and puncture. Compared with WT littermates, sepsis-induced mortality was improved in deficient mice, as well as in mosaic animals carrying both deficient and WT phagocyte subpopulations. In contrast, blood bacterial counts were greatest in deficient mice. Consistent with poor survival, WT mice also showed the most severe organ damage following sepsis. In mosaic animals, the deficient neutrophil subpopulations displayed increased organ recruitment and elevated CD11b membrane expression compared with WT neutrophil subpopulations within the same animal. The dynamics of sepsis-induced blood and organ cytokine content and WBC composition changes, including lymphocyte subsets in blood and bone marrow, showed differences among WT, deficient, and mosaic subjects, indicating that mosaic mice are not simply the average of the deficient and WT responses. Upon oxidative burst, interchange of oxidants between WT and deficient neutrophil subpopulations occurred in mosaic mice. This study suggests that mice mosaic for gp91phox expression have multiple advantages compared with WT and deficient mice during the septic course.     

Specificity of natural resistance to trematode infections in Biomphalaria glabrata

The 10-R2 strain of Biomphalaria glabrata was strongly resistant to various strains of Schistosoma mansoni in its laboratory of origin (NIH) and to three strains of S. mansoni we tested against it. However, subsequent development of three inbred lines of B. glabrata 10-R2 snails, separately maintained in our San Francisco laboratory, showed slight loss of resistance in one colony, very much less resistance (or partial susceptibility) in another, and retention of the original resistance in a third to the Puerto Rico (PR-1) strain of S. mansoni. No selection for resistance to infection was involved in the breeding protocol for these 10-R2 lines, so the changes were apparently random ones that became established in the separate inbred substrains. In spite of their changed response to the PR-1 strain of S. mansoni, all three 10-R2 substrains retained only slightly diminished resistance to S. mansoni Lc-1 strain and an essentially undiminished resistance to irradiated Echinostoma lindoense, E. paraensei and E. liei sporocysts. This suggests that natural resistance to S. mansoni PR-1 in B. glabrata is specific, a response that differs from the host response to either S. mansoni Lc-1 or to the echinostomes.     

Enrichment of threonine content in Saccharomyces cerevisiae by pathway engineering

In a previous work, we have investigated the effect of amplifying individually the genes of the threonine biosynthetic pathway on threonine accumulation by yeast. Here, we present the results of the simultaneous amplification of these genes in strains with different genetic backgrounds. These strains carry a mutant HOM3-R2 allele (coding for a feedback-insensitive aspartate kinase), and/or a mutant cha1 allele that makes it defective in threonine degradation by the catabolic L-serine (L-threonine) deaminase. The results show that the amplification of the clustered genes affects threonine and homoserine accumulation only when it includes the HOM3 gene, or when combined with a HOM3-R2 mutation. Similarly, the cha1 mutation is only effective when a certain amount of threonine is reached. Threonine overproduction affects other cellular functions such as the accumulation of other amino acids, the cell growth and metabolite excretion, probably reflecting a redirection of the carbon flux in the central metabolism.     

Studies on the relationship between dimethylnitrosamine-demethylase activity and dimethylnitrosamine-dependent mutagenesis in Drosophila melanogaster

The relationship between dimethylnitrosamine (DMN) demethylase activity and DMN-induced mutagenesis was investigated in Drosophila melanogaster. The activity of DMN-demethylase was at least 10-fold greater in the Hikone-R strain than in three other Drosophila strains. However, the sex-linked recessive lethal (SLRL) mutations induced by DMN in the four strains differed by less than 2-fold. Several possibilities to explain the lack of correlation between DMN-demethylase activity and DMN-induced mutations were tested and eliminated. They include: (i) the presence of inhibitors of DMN-demethylase in extracts of low-activity strains, (ii) a sex bias in the Hikone-R strain in which the enzyme activity is confined to the females, (iii) the possibility that DMN treatment induces DMN-demethylase activity in the low-activity strains and (iv) the possibility that Hikone-R has a much more efficient DNA repair system than the other strains. The results are discussed in terms of what is known about the role of DMN-demethylase in the metabolic activation of DMN in other systems.     

Host reactions in Biomphalaria glabrata to Schistosoma mansoni miracidia, involving variations in parasite strains, numbers and sequence of exposures

M-line Biomphalaria glabrata snails are susceptible to Puerto Rican (PR-1) strain of Schistosoma mansoni, but are resistant to a St. Lucian (LC-1) strain. 10-R2 B. glabrata snails are resistant to both strains of S. mansoni. When 10-R2 snails were exposed repeatedly to PR-1 S. mansoni miracidia for 5 consecutive days, all of the sporocysts were encapsulated and destroyed by the snails. Thirty-four per cent of sporocysts examined in M-line snails with similar exposures were also degraded. In double concurrent infections of M-line B. glabrata with [³H]leucine-labeled and unlabeled PR-1 and Lc-1 S. mansoni, the incompatible Lc-1 miracidia were selectively attacked and destroyed. This destruction occurred irrespective of the sequence of exposure of the 2 strains of miracidia, and whether or not the miracidia were labeled. Successful superinfection of M-line B. glabrata with homologous S. mansoni miracidia was obtained at least 4 days after the primary exposure to the miracidia.     

The in vivo modulation of dopamine synthesis by calcium ions: influences on the calcium independent release

To investigate the contribution of the dopamine (DA) synthesis to both the calcium-dependent and the carrier-mediated, mechanisms of DA release in the striatum, anaesthetized rats were locally superfused in the striatum with a push–pull cannula supplied with an artificial CSF containing tritiated tyrosine. DA, dihydroxyphenylacetic acid (DOPAC) and their respective specific activity were measured in effluent and used to evaluate changes in the DA synthesizing rate. Excluding calcium ions from the CSF only partially reduced spontaneous DA release (70%) still leaving a possible carrier-mediated DA release. This effect was not additive with a local superfusion with 0.1 mM a-methyl-p-tyrosine, a blocker of DA synthesis, suggesting that synthesis could already be reduced by calcium-free superfusion. Local superfusion with 100 μM cadmium in the presence or not of calcium ions, increased the DA release (220 and 350%, respectively), simultaneously reducing DA synthesis. Local application of 1 μM calcium ionophore (A23187) was without effect on the basal release of DA but enhanced DA synthesis and increased the amphetamine-evoked and carrier-mediated amine release. We conclude that DA synthesis can be a modulatory process of the firing-independent and carrier-mediated amine release while it weakly affects the classical calcium-dependent release.     

组蛋白甲基化与去乙酰化对蛇足石杉内生真菌盘长孢状刺盘孢合成石杉碱甲的影响

以蛇足石杉Huperzia serrata内生真菌盘长孢状刺盘孢Cg01菌株为研究对象,利用PEG介导的同源重组转化体系,对Cg01组蛋白甲基化酶基因（histone methyltransferases,HMT）CgClr4和组蛋白去乙酰化酶基因（histone deacetylase,HDAC）CgClr3、CgSir2进行基因敲除与回补,并通过实时荧光定量PCR（RT-qPCR）检测了回补株中对应基因表达量以及高效液相色谱HPLC检测突变体菌株中石杉碱甲huperzine A（HupA）产量。结果显示3个基因敲除突变体菌株ΔCgClr4、ΔCgClr3、ΔCgSir2的HupA产量分别为255μg/L、270μg/L、244μg/L,与野生型菌株相比分别下降了21.3%、16.6%、24.7%。在基因回补突变体菌株ΔCgClr4/CgClr4、ΔCgClr3/CgClr3、ΔCgSir2/CgSir2中,相应回补基因表达均与野生型无显著性差异,其HupA产量分别为351.9μg/L、334.7μg/L、331μg/L,回补菌株的HupA产量回复到野生型水平。结果表明这3个基因均具有调控内生真菌盘长孢状刺盘孢Cg01合成HupA的作用,为研究蛇足石杉内生真菌中石杉碱甲的合成调控机制提供了理论基础和新的思路。     

Mass-spectrometry based minisequencing method for the rapid detection of drug resistance in Mycobacterium tuberculosis

A MALDI TOF MS based minisequencing method has been developed and applied for the analysis of rifampin (RIF)- and isoniazid (INH)-resistant M. tuberculosis strains. Eight genetic markers of RIF resistance-nucleotide polymorphisms located in RRDR of rpoB gene, and three of INH resistance including codon 315 of katG gene and − 8 and − 15 positions of the promoter region of fabG1-inhA operon were worked out. Based on the analysis of 100 M. tuberculosis strains collected from the Moscow region in 1997–2005 we deduced that 91% of RIF-resistant and 94% of INH-resistant strains can be identified using the technique suggested. The approach is rapid, reliable and allows to reveal the drug resistance of M. tuberculosis strains within 12 h after sample isolation.     

Insulin-like growth factor 1 receptors (IGF1-R) and IGF1 in human breast tumors

To appreciate the IGF1 sensitivity of breast tumors we detected IGF1-R with a biochemical assay (RRA). We then localized and quantified IGF1-R on frozen tissue sections by histo-autoradiographic analysis (HAA). In some cases, the IGF1 and IGF1-R mRNA expression were studied by Northern blot analysis. We also studied the IGF1 plasma concentration in primary breast cancers compared to controls. IGF1-R (RRA) were found in 87% (n = 297) of the breast cancers. The mean geometric value was 3.87% (specific binding as percentage of total radioactivity); we found a highly significant correlation between IGF1-R and ER on the one hand (P = 0.0001) and PgR on the other (P = 0.0001) (Spearman test). The presence of IGF1-R was associated with a better prognosis, either on relapse-free survival (actuarial analysis: P = 0.004; Cox analysis: P = 0.005) or overall survival (respectively P = 0.003; P = 0.005). The median duration of follow-up was 30 months. By Cox analysis IGF1-R was a better prognostic factor than ER and PgR. In a series of 77 cases of benign breast disease only 47% (36/77) were positive; the mean geometric level was 1.8%. The HAA IGF1-R quantification in 20 breast carcinomas and 12 cases of benign breast disease confirmed the RRA results and demonstrated that the labeling was localized on the epithelial component. In four breast cancers, we did not detect IGF1 mRNA; IGF1-R probe demonstrated two major mRNAs of 11 and 7 kB. Finally we found that IGF1 plasma level was higher in breast cancer patients than in healthy controls of the same age. These results show that IGF1 is implicated in breast cancer growth and suggest that anti-IGF1 treatment might be useful in human breast cancer: for this reason, we and others carried out a phase II clinical trial with somatostatin.     

Mice transgenic for exon 1 of Huntington's disease: properties of cholinergic and dopaminergic pre-synaptic function in the striatum

In Huntington's disease (HD), neuronal loss is most prominent in the striatum leading to emotional, cognitive and progressive motor dysfunction. The R6/2 mice, transgenic for exon 1 of the HD gene, develop a neurological phenotype with similarities to these features of HD. In striatal tissue, electrically evoked release of tritiated acetylcholine (ACh) and dopamine (DA) were compared in wild-type (WT) and R6/2 mice. In R6/2 mice, the evoked release of ACh, its M2 autoreceptor-mediated maximum inhibition and its dopamine D2 heteroreceptor-mediated maximum inhibition was diminished to 51%, 74% and 87% of controls, respectively. Also, the activities of choline acetyltransferase and of synaptosomal high-affinity choline uptake decreased progressively with age in these mice. In the DA release model, however, electrical stimulation elicited equal amounts of [3H]-DA both in WT and R6/2 mice. Moreover, high-affinity DA uptake into striatal slices was similar in WT and R6/2 mice. In order to confirm these findings in vivo, intrastriatal levels of extracellular DA were measured by intracerebral microdialysis in freely moving mice: striatal DA levels were found to be equal in WT and R6/2 mice. In conclusion, in the transgenic R6/2 mice changes occur mainly in striatal cholinergic neurones and their pre-synaptic modulation, but not in the dopaminergic afferent terminals. Whether similar events also contribute to the pathogenesis of HD in humans has to be established.