A study of the inhibition/promotion effects of sodium-copper chlorophyllin (SCC)-mediated mutagenesis in somatic cells of Drosophila.

Sodium-copper chlorophyllin (SCC), a copper-porphyrin complex, has been shown to act as an inhibitor as well as a promoter of DNA-damage induction by a variety of mutagens in several test systems. In order to investigate the basis of this dual effect, experiments were carried out to compare the influence of pretreatment with intact SCC and that of its constituents, the metal-free protoporphyrin (PP-IX) and copper as CuCl(2). The wing-spot test was employed to monitor mutational events in somatic cells of Drosophila melanogaster. Heterozygous mwh+/+flr(3) larvae were treated for 24h with SCC, PP-IX, CuCl(2) or sucrose. Following this treatment, one group of larvae were immediately allowed to feed on instant medium containing 0.5mM N-nitroso-N-ethylurea (ENU) dissolved in phosphate buffer to reach pH 6. The remaining larvae received treatment with ENU with a delay of 1, 2 or 3days (DTD). Results revealed an (a) overall inhibitory effect for 0-DTD and 1-DTD after pretreatment with SCC, (b) only in 0-DTD after PP-IX, and (c) in all DTDs after treatment with CuCl(2). These results provide evidence that the copper ion plays a central role in the antimutagenic effect of SCC, and for a sustained period of time. Pretreatment with SCC and PP-IX produced a promoter effect at 2-DTD and 3-DTD. The results could be explained as an effect of the accumulation of metal-free porphyrin following the dissociation of the copper-porphyrin complex (SCC), the copper-ion reaching proteins to form complexes and participated in anabolic pathways.

Evidence that low concentrations of chlorophyllin (CHLN) increase the genetic damage induced by gamma rays in somatic cells of Drosophila.

It was first demonstrated in Salmonella that higher and lower concentrations of chlorophyllin (CHLN) may have effects in opposite directions, higher doses inhibiting and lower doses promoting the mutagenic activity of certain tobacco-related nitrosamines. Previous work of our group demonstrated that CHLN may have both a promoter and an inhibitory effect on mutagenesis in Drosophila. The present paper reviews the evidence obtained in our laboratory using gamma rays as the mutagenic agent, that higher and lower pretreatment concentrations of CHLN are associated with inhibitory and promoting effects, respectively, as in Salmonella. Employing the wing spot test, 48h larvae were pretreated with various concentrations of CHLN from 0 to 69 mM and then treated with 10 Gy gamma rays. With the highest concentration of CHLN, an approximate 54% reduction in mutagenesis was observed. At 35 mM a remnant of this inhibitory effect was found in that a significant decrease was limited to the twin spot category. Evidence of promotion was first seen at 4.3mM CHLN, an effect which persisted for the remaining five lower concentrations, the most pronounced evidence of promotion being found at the four lowest concentrations, 0.03-1.1 mM CHLN. It should be noted that no evidence of genotoxicity was found for CHLN alone, an observation consistent with the several reports in the literature. The results are taken as strong evidence that pretreatment with low concentrations of CHLN promotes DNA damage induced by gamma rays in somatic cells of Drosophila.

Evidence suggesting that chlorophyllin (CHLN) may act as an inhibitor or a promoter of genetic damage induced by chromium(VI) oxide (CrO3) in somatic cells of Drosophila.

In Drosophila, 48h-old larvae were pretreated for 24h with chlorophyllin (CHLN) or sucrose and then treated with chromium(VI) oxide (CrO(3)) immediately following completion of the pretreatment period (0-day delay) or delayed 1, 2 or 3 days. The effects were scored in the wing spot test. After delays of 0 and 1 day, clear evidence of a protective effect of CHLN was found. Contrarily, after delays of 2 and 3 days, the results showed a reversal, i.e. CHLN-related events appeared more frequently than those in the sucrose control suggesting a promoting effect. It would appear prudent that CHLN be tested in a variety of situations in any given organism before decisions are reached regarding its inhibitor/promoter effects.

The effect of treating Drosophila females with chlorophyllin on the yield of dominant lethals recovered from irradiated sperm.

Evidence is presented that treating the Drosophila female with chlorophyllin (CHLN) has a marked effect on the yield of dominant lethals induced by the irradiation of sperm. The yield is significantly greater in the embryonic period (between the egg and the first instar) and is significantly reduced in postembryonic stages compared with a sucrose control.

Evidence that chlorophyllin (CHLN) may behave as an inhibitor or a promoter of radiation-induced genetic damage in somatic cells of drosophila.

Irradiation of 96h old Drosophila following a 24h pretreatment with 5% chlorophyllin (CHLN) was delayed 0-4 days. The antimutagenic effect of CHLN in somatic cells monitored by the wing spot test persisted for 3 days after completion of the pretreatment and appeared to terminate at a time corresponding to the cessation of mitotic divisions of wing anlagen cells. Within the same population of cells, CHLN demonstrated both an inhibitory effect as measured in mwh single spot classes, and contrarily, a promoting effect in the class of mwh/flr twin spots and to an extent in the class of large flr spots. The reason for the contrasting effects of CHLN remains to be determined.

Chlorophyllin [CHLN] and the mutagenicity of monofunctional alkylating agents in Drosophila: the action of CHLN need not include an influence on metabolic activation.

The effect of chlorophyllin (CHLN) on the mutagenicity of four monofunctional alkylating agents (MFAAs) was evaluated in the wing spot test in Drosophila. Three of the compounds are direct-acting (ethylnitrosamine (ENU), methylnitrosourea (MNU), and methylmethanesulfonate (MMS)) and one indirect-acting (diethylnitrosamine, DEN). Results indicate that the mutagenicity of all four compounds is strongly inhibited by CHLN. The findings are not in agreement with the conclusion of Romert et al. (1992) that CHLN has no effect on the mutagenicity of direct acting MFFAs inferred from their work with MNU and ethylmethanesulfonate (EMS) in the V79 and Salmonella in vitro test systems. The results suggest the possibility that the action of CHLN need not include an inhibiting effect on metabolic activation.

On the persistence of the radioprotective effect of chlorophyllin (CHLN) in somatic cells of Drosophila.

By delaying the time of gamma irradiation of 72 h larvae, pretreated at 48 h with 5% chlorophyllin (CHLN), it was established that the overall inhibiting effect of CHLN in somatic cells of Drosophila, as measured in the wing spot test, persists for about 4 days or until the time of cessation of the proliferation of wing anlagen. In the same population of cells, some spot classes gave evidence of an inhibitory effect whereas others did not arguing against the suggestion that the radioprotective effect of CHLN is a consequence of an induced delay in development, shrinking of the potential radiation target and lowering the probability of induced events. Other observations of interest are described.

On the recovery of single spots with the flr phenotype in the wing spot test in Drosophila.

Graf et al. (U. Graf, F.E. Würgler, A.J. Katz, H. Frei, H. Juon, C.B. Hall, P.G. Kale, Somatic mutation and recombination test in Drosophila, Environment Mutagen. 6 (1984) 153-188.) described the overall results of assays of a series of compounds in the Drosophila wing spot test as indicating that single mwh spots appeared most frequently, followed by less frequent twin spots with both mwh and flr cells and lastly the 'quite rare' single flr spots. Data are presented below demonstrating that some compounds behave in a manner consistent with the above description, whereas others do not in that the frequency of single flr spots is equal to or exceeds that of twin spots and cannot be described as occurring 'rarely'. It is suggested that (large) flr singles be used as a measure of mutations/deletions directly from treated transheterozygotes. An argument is presented questioning the use of treated mwh +/+ TM3 individuals as an assay of mutations/deletions at the mwh+ locus.

Evidence for an effect of exposure to low levels of alpha particle irradiation in larval cells of Drosophila as measured in the wing-spot test.

Larvae of Drosophila were exposed to a range of concentrations of alpha particles from 3 to 318 mRad, and genetic effects measured in the wing-spot test. The results were positive, and evidence of a linear relationship between exposure and response observed. The induction of chromosome breakage is suggested by the significantly higher frequency of twin spots in the treated series compared with controls.

Chemical mutagenesis testing in Drosophila. IX. Results of 50 coded compounds tested for the National Toxicology Program.

Fifty chemicals were tested for mutagenic activity in post-meiotic and meiotic germ cells of male Drosophila melanogaster using the sex-linked recessive lethal (SLRL) assay. As in the previous studies in this series, feeding was chosen as the first route of administration. If the compound failed to induce mutations by this route, injection exposure was used. One gaseous chemical (1,3-butadiene) was tested only by inhalation. Those chemicals that were mutagenic in the sex-linked recessive lethal assay were further tested for the ability to induce reciprocal translocations. Eleven of the 50 chemicals tested were mutagenic in the SLRL assay. These included bis(2-chloroethyl) ether, 1,4-butanediol diglycidyl ether, 1-chloro-2-propanol, dimethyl methylphosphonate, dimethyl morpholinophosphoramidate, dimethyloldihydroxyethylene urea, 2,2-dimethyl vinyl chloride, hexamethylphosphoramide, isatin-5-sulfonic acid (Na salt), isopropyl glycidyl ether, and urethane. Five of these, including 1,4-butanediol diglycidyl ether, 2,2-dimethyl vinyl chloride, hexamethylphosphoramide, isopropyl glycidyl ether, and urethane, also induced reciprocal translocations.

Chemical mutagenesis testing in Drosophila. X. Results of 70 coded chemicals tested for the National Toxicology Program.

Seventy chemicals were tested for the ability to induce sex-linked recessive lethal (SLRL) mutations in postmeiotic and meiotic germ cells of male Drosophila melanogaster. As in the previous studies in this series, adult feeding was chosen as the first route of administration. If the compound failed to induce mutations by this route, injection exposure was used. Two chemicals, n-butane and propylene, were gaseous and therefore tested only by inhalation. One chemical (dimethylcarbamoyl chloride) was tested only by injection. Those chemicals that were mutagenic in the SLRL assay were further tested for the ability to induce reciprocal translocations. Sixteen of the 70 chemicals tested were mutagenic in the SLRL assay: 3-chloro-2-methylpropene, 3-(chloromethyl)pyridine HCl, dimethylcarbamoyl chloride, HC blue 1,3-iodo-1,2-propanediol, malaoxon, N,N'-methylene-bis-acrylamide, 4,4'-methylenedianiline 2HCl, ziram, cis-dichlorodiaminoplatinum II, 1,2-dibromoethane, dibromomannitol, 1,2-epoxypropane, glycidol, myleran, and toluene diisocyanate. The last seven also induced reciprocal translocations. A comparison of the results from the SLRL assay with other assays for mutagens and carcinogens suggests that the SLRL assay is highly specific, but poorly sensitive, both for mutagens and potential carcinogens.

Evaluation in Drosophila melanogaster of the mutagenic potential of furfural in the mei-9a test for chromosome loss in germ-line cells and the wing spot test for mutational activity in somatic cells.

The mutagenic potential of furfural was evaluated by means of the chromosome loss test in germ cells and the wing spot test in somatic cells of Drosophila melanogaster. The chromosome loss test was carried out employing repair-proficient as well as repair-deficient females. Males carried the compound Y chromosome, BSYy+. Two routes of administration were used: injection and feeding of adult males. Genetic damage was demonstrable after matings of treated males with females carrying the excision repair-deficient mutant mei-9a. The somatic mutation and recombination test was carried out treating 72-h transheterozygous mwh+/+flr3 larvae. Acute treatment of larvae was chosen as the method of exposure. Evidence indicates that furfural induces somatic damage as measured in the wing spot test.

Chemical mutagenesis testing in Drosophila: VIII. Reexamination of equivocal results.

Twelve percent of the chemicals tested for mutagenicity by the National Toxicology Program (NTP) using the Drosophila sex-linked recessive lethal assay have been classified as producing equivocal results. We have reexamined the published data and the criteria used to determine mutagenicity in light of the historical distribution of the concurrent negative controls for this project. Many of the chemicals that originally produced equivocal results have been retested under code. As a result of changes to incorporate a comparison with the historical control in the algorithm used to determine mutagenicity and as a result of new data accumulated, 4 of the 25 chemicals that gave equivocal results are judged to be mutagenic, and 11 others are judged to be nonmutagenic under our test conditions.

Aneuploidy in Drosophila, II. Further validation of the FIX and ZESTE genetic test systems employing female Drosophila melanogaster.

Two sensitive genetic systems for the detection of germline aneuploidy employing Drosophila melanogaster females were described in the first paper of this series (Zimmering et al., submitted to Mutation Research). Designated FIX and ZESTE, these systems permit the rapid and efficient detection of exceptional offspring derived from aneuploid female germ cells. The current report presents test results from a survey of 8 additional chemicals that have been analyzed in both systems. The tested chemicals include: acetonitrile, cadmium chloride, carbendazim, dimethylsulfoxide (DMSO), methylmercury(II) chloride, methoxyethyl acetate, propionitrile and water. Excluding the negative control, water, only the fungicide carbendazim failed to induce aneuploidy in either test system. Of the remaining 6 chemicals one, methylmercury(II) chloride, was positive in the FIX system but not in ZESTE, while MEA was positive in ZESTE and borderline in FIX. The results provide little evidence of germ-cell stage specificity of response to the tested chemicals. Comparison of the induced rates of aneuploidy i indicates that these can exhibit departures from simple additivity to the spontaneous rates: induced rates in the ZESTE system are generally higher and more variable than those from FIX. Possible reasons for the difference in responsiveness between FIX and ZESTE flies are discussed as is the question of the classification of those chemicals which induce chromosome loss events but not chromosome gains.

Aneuploidy in Drosophila, IV. Inhalation studies on the induction of aneuploidy by nitriles.

The Drosophila ZESTE system was used to monitor the induction of sex chromosome aneuploidy following inhalation exposure of adult females to four nitriles: acetonitrile, propionitrile, acrylonitrile and fumaronitrile. Acetonitrile and propionitrile were highly effective aneuploidogens, inducing both chromosome loss and chromosome gain following brief exposures to low concentrations of these chemicals, and these nitriles also induced rapid paralysis. Acrylonitrile-induced chromosome loss only but did not induce paralysis. Fumaronitrile, in contrast with the results reported in yeast, was ineffective in inducing chromosome loss or gain. Virtually all exceptional offspring induced by acetonitrile and propionitrile were recovered in the first sampled eggs, corresponding to treated mature oocytes. Additionally, the time interval between treatment and sampling was shown to be important, suggesting rapid loss or detoxification of the nitriles. Genetic analysis demonstrated that most aneuploids resulted from induced segregation errors during the first division of meiosis. Cold treatments were found to be ineffective in enhancing the effects of acetonitrile, suggesting important differences between the Drosophila and yeast aneuploidy detection systems. Possible mechanisms by which nitriles may disrupt chromosome segregation in Drosophila oocytes are considered.