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E L S E V I E R Mutation Research 324 (1994) 23-27 Mutation Research Letters

Refractory ceramic fibers (RCFs) induce germline aneuploidy in Drosophila oocytes

Christopher J. Osgood *

Department of Biological Sciences, Old Dominion University, Norfolk, VA 23529, USA

(Received 17 May 1993; revision received 2 February 1994; accepted 7 February 1994)

Abstract

Mineral fibers are ubiquitous in the natural environment and are widely used in industry in such applications as insulators. We have previously shown that asbestos fibers induce aneuploidy in oocytes of Drosophila melanogaster; here we extend those studies by testing refractory ceramic fibers (RCFs) for their mutagenicity to germ cells. The results establish that the tested RCFs are inducers of aneuploidy following feeding of adult females. A subset of the RCFs were also effective following larval feeding. Our results suggest that RCFs, like certain asbestos fibers, may pose a hazard to germ cells.

Key words: Aneuploidy; Refractory ceramic fibers; Drosophila melanogaster

I. Introduction

We have previously shown that, following feed- ing of adult Drosophila females, asbestos fibers induce germline aneuploidy in oocytes (Osgood and Sterling, 1991). Asbestos fibers are composed largely of silica and the available evidence sug- gests that those fiber types that are long and thin in shape are the most hazardous to mammalian cells (Hesterberg et al., 1984; Mossman et al., 1990). Experiments with silica glass fibers (GFs) indicate that these too are mutagenically active, including the induction of aneuploidy, when ex- posed to cultured mammalian cells (Hesterberg et al., 1984). Also, as in the results with asbestos,

* Corresponding author.

extended rod-like GFs were the most effective forms; indeed, some GFs were as effective as asbestos in inducing transformation of mam- malian cells in vitro (Oshimura et al., 1984).

Refractory ceramic fibers (RCFs) are widely used in industry as insulating materials (Friar et al., 1989). RCFs are similar in chemical composi- tion and physical dimension to asbestos fibers. This prompted us to ask whether these fibers are similarly effective as inducers of germline aneu- ploidy following treatment of Drosophila females. The data reported here include results from adult and larval feeding on RCFs. Water, or sucrose water, was used as a negative control, while chrysotile asbestos was used as a positive fiber control (Osgood and Sterling, 1991). The results indicate that RCFs, similar to chrysotile asbestos,

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24 C.J. Osgood /Mutation Research 324 (1994) 23-27

Table 1 Mineral fibers tested for induction of aneuploidy in Drosophila

Fiber D x L Components > 10% of total

RCF-1 1.1 x 22 SiO 2 (48); AI203 (48) RCF-2 1 x 18.8 SiO 2 (50); AI203 (35); ZrO 2 (15) RCF-3 1.2 X 25 SiO 2 (51); AI203 (49) RCF-4 a 1.4 X 9.8 SiO 2 (48); AI203 (48) Chrysotile 0.08 X 0.99 SiO 2 (42); MgO (43)

a RCF-4 is the "after service" form of RCF-2. Listed are the fiber types tested in these experiments, their dimensions in microns (diameter (D)x length (L)), and their percent compositions (percentage of each molecule in brack- ets).

induce germline aneuploidy. The possibility that many silica based fibers may be mutagenic to germ cells is discussed.

2. Materials and methods

Chemicals. The RCFs and chrysotile asbestos were the generous gift of the Mountain Technical Center, Littleton, Colorado. The physical charac- teristics of the tested fibers are summarized in Table 1.

Treatments. For the adult feeding experiments, fibers were prepared at 25 m g / m l in 5% su- crose-water . All fibers formed thick slurries rather than true solutions and were therefore difficult to pipet evenly onto the feeding surfaces. 2.5-ml aliquots of the fiber slurries were dis- pensed onto 4.7-cm glass-fiber filters placed in the bottom of pint glass bottles. Females were transferred into these feeding bottles, 50 females per bottle. The flies were transferred to freshly prepared feeding bottles after 24 and 48 h. At the end of 3 days' feeding (72 h) the females were removed and placed in empty culture bottles. During chemical feeding, the flies showed no obvious aversion to fiber slurries and appeared to feed directly on the fibers. As was the case previ- ously for tested asbestos fibers (Osgood and Ster- ling, 1991), no toxicity was observed following the 3-day feeding on the RCFs or on chrysotile. At the end of the feeding period, the females were

mated and their offspring tested as described below.

In larval feeding experiments, treatment oc- curred throughout larval development as follows. Matings of 20 pairs of P0 males and females (genetic composition described below) were set on standard medium for 24 h. The same animals were then directly transferred to a second set of bottles supplemented with the test fibers. These latter bottles were prepared by adding weighed amounts of fiber to Drosophila Instant Medium (Carolina Biological Supply, NC), mixing, and then addition of an equal volume of water (50 ml). The medium was allowed to harden for 15 min before transfer of the P0 adults. The flies were allowed to lay eggs for 48 h in the treatment bottles and were then cleared. Bottles were kept at 25°C; no differences were observed in develop- ment rates between the fiber treated and water control bottles. 10-17 days after initiation of the cultures, the P1 adults emerged and automatic virgin females were collected and used to set the P1 test mating as described below.

Genetic crosses. The ZESTE test methodology has been described in detail previously (Zimmer- ing et al., 1990; Osgood et al., 1991). In brief, test females, obtained as automatic virgins, are de- rived from a cross of P0 females (y z / y z; spa r"l) with P0 males (y2 z f . y L / y S , y+; spaPOl), and are of the genotype y z / y 2 z f . y L ; spaPOl (zeste- eyed females). Such females, t reated or not, were small mass-mated with attached X - Y males, X.Y, w/O; net, approximately 20 pairs per culture bot- tle (for further information on genetic symbols, see Lindsley and Zimm, 1992).

In these experiments, 3 germ-cell samplings, or broods, were collected from treated or control females and designated Broods A, B and C. These were of 2-day, 2-day and 3-day duration, respec- tively.

Emerging offspring, the Fls, were counted and examined for eye colors. Regular offspring from the P1 mating are, with respect to eye color, phenotypically wild-type females and males (Zimmering et al., 1990). Exceptional offspring, arising from aneuploid ooeytes, were of two main types: (1) zeste-eyed females, arising from the fertilization of diplo-X eggs by nullo-X,Y sperm;

C.J. Osgood / Mutation Research 324 (1994) 23-27

Table 2 Aneuploidy induced following feeding of Drosophila adult females on RCFs

25

Brood A Brood B Brood C Sum (A-C)

N CL CG N CL CG N CL CG N CL CG (CL + CG)

Control 4 400 1 0 6 508 0 0 4 782 2 0 15 690 3 0 3 (0.02%)

RCF-1 11458 1 2 13543 4 3 11423 7 2 36424 12 7 * 19 * (0.052%)

RCF-2 12928 2 0 12684 9 2 12796 14 2 38408 25 * 4 29 * (0.076%)

RCF-3 9683 2 0 11363 3 0 11249 13 2 32295 18 * 2 20 * (0.062%)

RCF-4 13229 2 1 16159 6 1 13442 13 1 42830 21 * 3 24 * (0.056%)

Chrysotile 6 685 4 4 7 376 1 0 6 759 5 0 20 820 10 4 * 14 * (0.070%)

Symbols: N, total number of offspring; CL, chromosome loss; CG, chromosome gain; * Statistically significant difference from the control ( P < 0.05).

such females are referred to as arising from chro- mosome gain (CG); and (2) white-eyed males, arising from the fertilization of nullo-X eggs by X.Y bearing sperm; these are inferred to arise from chromosome loss (CL) of one of the mater-

nal X chromosomes. All bona fide exceptional offspring are non-spa p°~ and non-net.

Frequencies of exceptional offspring were cal- culated by dividing the number of CL, CG or both types of exception by the total number of

Table 3 Aneuploidy induced following feeding of Drosophila female larvae on RCFs

Brood A Brood B Brood C Sum (A-C)

N CL CG N CL CG N CL CG N CL CG (CL + CG)

Control 18481 3 0 20387 4 0 20644 2 0 59512 9 0 9 (0.015%)

RCF-1 6132 6 0 5954 7 1 6094 2 2 18180 15 ** 3 * 18 ** (0.25 g) (0.099%)

RCF-2 5 243 0 0 8151 2 2 8071 1 0 21465 3 2 5 (0.25 g) (0.023%)

RCF-2 4 567 0 0 6 068 1 0 5175 0 0 15 810 1 0 1 (1.0 g) (0.006%)

RCF-3 6828 2 2 9983 3 3 9699 8 3 26510 13 * 8 * 21 ** (0.25 g) (0.079%)

RCF-4 4 973 1 0 4 327 0 2 4 341 0 0 13 641 1 2 3 (0.25 g) (0.021%)

RCF-4 5 296 2 0 3 683 0 1 3 759 0 0 12 738 2 1 3 (1.0 g) (0.024%)

Chrysotile 6 262 2 1 5 372 0 0 5 193 2 0 16 827 4 1 5 (0.25 g) (0.030%)

Symbols as in Table 2. ** P value of comparison with control < 10 -6.

2 6 C.J. Osgood / Mutation Research 324 (1994) 23-27

Fls scored. Statistical analyses were carried out as described previously (Margolin et al., 1983; Osgood et al., 1991).

Results

Concurrent controls were carried out for each of the AF and LF experiments reported here. No evidence for statistically significant heterogeneity was detected among the controls, and these were accordingly pooled in Tables 2 and 3 and for statistical analysis. The treated results come from single experiments carried out at the indicated dose of fibers.

The adult feeding data are presented in Table

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A. A d u l t l e e d l n g (25 m g / m l )

B . Larval feeding (250 rngro t l )

C O N C H R Y S

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Fig. 1. RCF-induced aneuploidy. Pooled percents of excep- tional offspring (CL+CG) from Tables 2 and 3 are plotted. Statistically significant differences between treated and con- trol are marked by asterisks. Error bars represent standard errors of the means calculated as described in Margolin et al. (1983). Abbreviations: CON, control; CHRYS, chrysotile as- bestos; RCF, refractory ceramic fiber.

2, the larval feeding data in Table 3. A graphic summary of the adult and larval results is shown in Fig. 1.

Following adult feeding, all of the tested fibers induced statistically significant frequencies of ex- ceptional offspring, and in general, the induced rates were 2-3-fold higher than the control rate. As found previously (Osgood and Sterling, 1991), chrysotile asbestos was also effective in induction of aneuploidy, and was thereby validated as our positive control. Of the tested fibers, RCF-1 and chrysotile were positive for the induction of chro- mosome gain (CG); all others induced chromo- some loss (CL) only. RCF-4 is the after service (i.e., furnace-heated) version of RCF-2; the re- sults from these two fibers did not differ statisti- cally in the aneuploidy assays.

After larval feeding, somewhat different re- sults were obtained. Only fibers RCF-1 and RCF- 3 were effective inducers of aneuploidy (Table 3). Fibers negative at the initially tested dose of 250 mg per bottle were subsequently tested on the higher dose of 1 g fiber per bottle; again, no significant effect was detected for RCFs 2 and 4. Both RCF-1 and RCF-3 induced significant levels of both chromosome gain and loss, although nu- merically there were more chromosome losses recovered for each fiber. The results suggest that RCF-3 was more effective as an inducer of CG than was RCF-1 (0.030% vs. 0.017% CGs, respec- tively), but the apparent difference was not statis- tically significant.

Apparent differences in aneuploids recovered were observed over broods. For example, in the adult feeding experiments, for each of the tested RCFs more exceptional offspring were recovered in Brood C, a trend which is most dramatic for RCF-3 where approximately 75% of the excep- tions were recovered in Brood C. The results with the RCFs are in apparent contrast with those from chrysotile: the asbestos fiber induced nearly equivalent yields of exceptional offspring in all broods. The larval feeding experiments give no clear evidence of brood-specific patterns of re- sponse. In view of our limited experience with mineral fiber compounds, we are reluctant at this point to attempt definitive conclusions regarding apparent brood differences. Further experiments

CJ. Osgood / Mutation Research 324 (1994) 23-27 27

would be required before clear patterns could be 1984), but also because of potential damage to established with certainty, their germ cells.

4. Discussion

The results reported here establish that, as in the case of previously tested chrysotile and amosite asbestos (Osgood and Sterling, 1991), RCFs induce sex chromosome aneuploidy when fed to adult Drosophila females. The observed effects are modest but statistically significant, and the rates of aneuploidy induced by the RCFs are in the range previously found for chrysotile and amosite asbestos (Osgood and Sterling, 1991; and this report). RCF-1 is potentially of greatest in- terest since it, like chrysotile, induces significant increments in chromosome gain. As we have dis- cussed previously (Osgood et al., 1991), agents that induce CG as well as CL are candidate spindle poisons since failures of the spindle dur- ing cell division should generate CGs as often as CLs. On the other hand, chemicals that induce CL only may simply be causing chromosome breakage. In this context, it is interesting to note that non-fibrous minerals are generally less toxic and induce primarily chromosome fragmentation (Oshimura et al., 1984).

The larval feeding results identified as positive only a subset of the tested fibers, namely RCF-1 and RCF-3. We found this result surprising since larvae were expected to be better able to con- sume insoluble materials, including the fiber slur- ries. We must note, however, that we have no measure of actual consumption in these experi- ments so it is conceivable that larvae in fact received a lower dose than adults. Alternatively, there may be unidentified differences in the sen- sitivities of the larval and adult gonads that affect their response to the fibers.

Our results suggest that, at least for Drosophila female germ cells, RCFs may pose a threat as inducers of aneuploidy. To the extent that results from Drosophila can be extrapolated to higher organisms, some concern may be appropriate for workers who are exposed to high levels of mineral fibers, not only because of their risk for develop- ing somatic cancers (Hesterberg and Barrett,

Acknowledgements

Experiments supported in part by a grant from the March of Dimes. We thank Mr. Thomas June of the Mountain Technical Center for supplying the mineral fibers as well as technical information on those fibers. Ms. Jennifer Wohl provided ex- cellent technical assistance in the execution of the experiments reported here.

References

Friar, J.J., and A.M. Phillips (1989) Exposure to ceramic man-made mineral fibres, in: J. Bignon, J. Peto and R. Saracci (Eds.), Non-occupational Exposure to Mineral Fi- bres, IARC Publ. No. 90, Lyon, France.

Hesterberg, T.W., and J.C. Barrett (1984) Dependence of asbestos- and mineral dust-induced transformation of mammalian cells in culture on fiber dimension, Cancer Res., 44, 2170-2180.

Lindsley, D.L., and G.G. Zimm (1992) The Genome of Drosophila melanogaster, Academic Press, San Diego.

Margolin, B.H., B.J. Collings and J.M. Mason (1983) Statisti- cal analysis and sample size determinations for mutagenic- ity experiments with binomial responses, Environ. Muta- gen., 5, 705-716.

Mossman, B.T., J. Bignon, M. Corn, A. Seaton and J.B.L. Gee (1990) Asbestos: Scientific developments and implications for public policy, Science, 247, 294-301.

Osgood, C., and D. Sterling (1991) Chrysotile and amosite asbestos induce germline aneuploidy in Drosophila, Muta- tion Res., 261, 9-13.

Osgood, C., S. Zimmering and J.M. Mason (1991) Aneuploidy in Drosophila, II. Further validation of the FIX and ZESTE genetic test systems employing female Drosophila melanogaster, Mutation Res., 259, 147-163.

Oshimura, M., T.W. Hesterberg, T. Tsutsui and J.C. Barrett (1984) Correlation of asbestos-induced cytogenetic effects with cell transformation of Syrian hamster ovary cells in culture, Cancer Res., 44, 5017-5022.

Peto, J. (1989) Fibre carcinogenesis and environmental haz- ards, in: J. Bignon, J. Peto and R. Saracci (Eds.), Non-oc- cupational Exposure to Mineral Fibres, IARC Publ. No. 90, Lyon, France.

Zimmering, S., C. Osgood and J.M. Mason (1990) Aneuploidy in Drosophila, I. Genetic test systems in the female Drosophila melanogaster for the rapid detection of chemi- cally induced chromosome gain and loss, Mutation Res., 234, 319-326.

Communicated by M.D. Shelby