Mutation Research, 251 (1991) 91-97 © 1991 Elsevier Science Publishers B.V. All rights reserved 0027-5107/91/$03.50 ADONIS 002751079100218S

91

MUT 05011

Low-level chemiluminescence from Drosophila melanogaster fed with chemical mutagens polycyclic aromatic hydrocarbon quinones and a carcinogenic bracken fern Tomoko Sato 1, Humio Inaba 1,2 Kazuaki Kawai 3, Hideyuki Furukawa 3, Iwao Hirono 4 and Teruo Miyazawa 5 1 Research Development Corporation of Japan, Biophoton Project, c / o Kohjinkai 2-1-6, Tsutsujigaoka Miyagino-ku, Sendai 980, 2 Research Institute of Electrical Communication, Tohoku University, Katahira Aoba-ku, Sendai 980, 3 Laboratory of Biological Science, Faculty of Pharmacy, Meijo University, Tempaku-ku, Nagoya 468, 4 Department of Pathology, School of Medicine, Fujita Health University, Dengaku-ku, Toyoake 442 and s Department of Food Chemistry, Tohoku University, Tsutsumidori Aoba-ku, Sendai 980 (Japan) (Received 26 March 1990) (Revision received 18 June 1990) (Accepted 22 April 1991)

Keywords: Drosophila; Chemiluminescence; Polycyclic aromatic hydrocarbon; Bracken fern; Lipid peroxide; Mutagen

Summary The spontaneous photon emission (chemiluminescence) from Drosophila melanogaster fed chemical mutagens, polycyclic aromatic hydrocarbon quinones, and a carcinogenic bracken fern was studied. The fly chemiluminescence was evidently enhanced by mutagen or carcinogen administration and was increased proportionally to the administered amount of tested compound. Strong chemiluminescence was observed especially at the larval stage. Living larvae emitted stronger chemiluminescence than their homogenate. The chemiluminescence from Drosophila melanogaster fed polycyclic aromatic hydrocarbon quinones showed a linear relation with the mutation frequency in the Drosophila wing spot test. The chemiluminescence from flies fed a bracken fern decreased by the addition of free radical scavengers and active oxygen quenchers. The phosphatidylcholine hydroperoxide concentration in the flies was increased proportionally with the chemiluminescence intensity. It seems that the free radical formation is stimulated as shown by the enhanced chemiluminescence in mutagen- or carcinogen-dosed flies, and as a result, lipid peroxide accumulation accompanies mutation in Drosophila melanogaster.

Low-level chemiluminescence (CL) is known to be associated with oxidation reactions involving molecular oxygen (Vassil'ev and Vichutinskii,

Correspondence: Dr. T. Miyazawa, Department of Food Chemistry, Tohoku University, Tsutsumidori Aoba-ku, Sendal 980 (Japan).

1962). In biological systems, it is suggested that free radicals, active oxygens and lipid peroxides are involved in the metabolic processes of mutagens and carcinogens. Although several studies have been carried out such as the CL of tumor tissues (Boveris et al., 1985), the CL originating in carcinogen metabolism in in vitro systems (Ham, man and Seliger, 1976; Hamman et al., 1981;

92 Emerole and Dixon, 1980; Seliger et al., 1982) and the CL dependent on carcinogenesis promoters (Fischer and Adams, 1985; Fischer et al., 1985), there has been no direct measurement of the low-level CL generated in living whole bodies, especially for in vivo systems. Drosophila melanogaster, the fruit fly, is a higher animal that has an enzymic system like rats and mice for metabolizing drugs. Therefore, the fly has often been used for mutation research. Recently, the Drosophila wing-spot test has been developed for mutagen screening as a convenient in vivo assay (Graf et al., 1984). In the present paper, we report the spontaneous Drosophila melanogaster CL observed on administering chemical mutagens, polycyclic aromatic hydrocarbon (PAl-/) quinones, and a bracken fern known to contain the carcinogen ptaquiloside (Evans and Mason, 1965; Hirono et al., 1984), and discuss the free radical production and the tissue lipid peroxidation in the process of mutation expression. Material and methods

Fly strains and treatment with chemicals The female stock y;mwh jr, spa p°! and the male stock y;flr3/TM3,Ser were used. From crosses between these stocks, we obtained larvae transheterozygous for the locus mwh (multiple wing hairs) and the locus fir (flare hairs), which are on the left arm of chromosome 3 (Graf et al., 1984). Flies from the 2 stocks were introduced at a ratio of 20 virgin females that emerged within 8 h to 20 males in culture bottles (5 cmZx 10 cm) containing 10 ml of medium (consisting of agar 2.3 g, glucose 30 g, corn meal 23 g, dry yeast (Ebios) 12 g, propionic acid 1.2 ml and water 300 ml) (Fujikawa, 1988). The parental flies were discarded 24 h after mating and oviposition. Two or three days later, larvae from 60 + 36 h after egg laying were collected. The larvae were washed with 20% sucrose solution and then transferred to 'treatment' vials containing 1.2 g of dry instant medium (Carolina Biological Supply Co., Burlington, NC) dissolved in 4 ml of water containing a PAH solution. The solution was prepared by dissolving 1 mg of a test PAH in 200/zl of ethanol. The bracken fern was mixed as a powder. The

larvae were given 10% or 34% bracken-containing medium. They were kept in the 'treatment' vials at 25 °C and 60% relative humidity. Control flies were cultured under the same conditions with omission of the test mutagen. Phenobarbital (0.1%, Wako Pure Chem. Ind., Osaka) was mixed in the medium 2 days before test mutagen administration.

Chemicals 1,6-Pyrenequinone (1,6-Pq), 1,8-pyrenequinone (1,8-Pq), 1,6-benzo[a]pyrenequinone (1,6-BPq), and 3,6-benzo[a]pyrenequinone (3,6-BPq) were obtained by photooxidation and photopolymerization of air dust particles that were adsorbed on filter paper. They were identified by mass spectrography and NMR spectrography, substituted for nitrogen gas and sealed up, and then stored at - 8 0 ° C. The preparation of these chemicals has been described in detail by Furukawa et al. (1989). Pyrene and anthracene were purchased from Tokyo Chem. Ind. Co., Tokyo. The bracken fern was collected in Hokkaido, in the northern part of Japan, in August, and was dried and powdered (Hirono et al., 1984).

Drosophila wing spot test Flies (mwh + / + f i r ) were preserved in 70% ethanol, and afterwards used for scoring mutant colonies. Wings were mounted on glass slides with Faure's solution (gum arabic 30 g, glycerol 20 ml, chloral hydrate 50 g, and water 285 ml, purchased from Wako Pure Chem. Ind., Osaka) and scored under a microscope at 400-fold magnification for clones with mutant hairs. The number of spots was recorded. The criteria adopted for scoring have been described by Clements et al. (1988).

Measurement of chemiluminescence A synchronous single photon counting apparatus (manufactured as type CLD-100 by Tohoku Electronic Ind. Co., Sendai) was provided by the Biophoton Project of the Research Development Corporation of Japan. A Hamamatsu R1333 photomultiplier tube with a spectral response range of 300-900 nm was used and kept at - 2 0 ° C using a thermoelectronic cooler to minimize background counts. Phototube output was sent to

93 sample cell [~lsampte room ~ -heater sample

~ ~ ~

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preamplifier dark room high v o l t a g e [

power

supplyj

F

I

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I

[ I

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Fig. 1. Schematic diagram of the synchronous single photon counting apparatus.

an amplifier-discriminator and connected to a microcomputer for data management (Fig. 1) (Inaba et al., 1982). Living larvae (5-8 days old) were cleaned twice with 20% sucrose solution, living pupae (6-13 days old) were cleaned twice with physiological saline (pH 7.0), and living adults (0-5 days after emergence) were cooled to 4 ° C . Fly homogenates were prepared in a ratio of 0.1 g of fly to 4 ml of physiological saline (PBS) using a teflon homogenizer under ice-cold conditions. A fly sample was placed in a glass plate (4.5 cm in diameter and 1.5 cm in height) and the CL was measured at 25 °C or 30 °C in the sample room for 25 or 30 min. CL intensity is expressed in terms of average counts per minute and corrected for background counts (the plate blank 617 cpm, the PBS blank 120 cpm).

Quenching test The chemicals were obtained from the following sources: L-ascorbic acid (Wako Pure Chem. Ind., Osaka); glutathione and catalase (Sigma Chem. Co., St. Louis, M O ) ; Tiron (C6H 4Na2OsS 2) (Dojin Chem. Inst., Kumamoto); superoxide dismutase (SOD) (Toyobo Co., Osaka). They were dissolved in re-distilled water. 4 ml homogenate from adult flies fed 10% bracken fern was diluted with 12 ml PBS. The CL of a 3-ml sample was measured for 20 min. After addition of 30-300 /xl of radical scavenger solutions, the CL was again measured for 20 rain, and the CLs before and after the addition of chemicals were compared.

PCOOH concentration Determination of phosphatidylcholine hydroperoxide (PCOOH) in fly lipids was performed according to the chemiluminescenceHPLC (CL-HPLC) method of Miyazawa (1989). The fly total lipid was extracted quantitatively with a mixture of chloroform and methanol (2 : 1, v/v, containing 0.002% butyl hydroxytoluene as antioxidant). The extraction was repeated 3 times. After dehydration and drying of the combined chloroform layer, the total fly lipid was diluted with 40 /.d of chloroform-methanol (2:1, v/v.), and a 20-/zl portion was subjected to the CLHPLC analysis. Calibralion was performed with authentic P C O O H prepared from egg yolk phosphatidylcholine (PC), as reported previously (Miyazawa et al., 1988a). The hydroperoxide concentration is expressed as pmole hydroperoxideO 2. Results

Fig. 2 shows the CL intensity from Drosophila melanogaster larvae fed with chemical mutagen 1,6-BPq (0.02%). Phenobarbital treatment was performed in order to activate the drug-metabolizing enzymes. The CL intensity of the 1,6-BPqfed living larvae was 3 times stronger than that of the control larvae which had not received 1,6-BPq. The phenobarbital treatment had no effect on the CL of living whole larvae. The CL intensity of

r

living intact

I

hromOgenized 4rnl PBS

I

6000 I E

c -~ c~ 4000 E~ =o

~: ~ v~ 2000

control

ii

1,6-BPq led

control

1,6-BPq fed

Fig. 2. Chemiluminescencefrom Drosophilamelanogaster fed 1,6-benzo[a]pyrenequinone(1,6-BPq).The white bars indicate the 0.1% phenobarbital-treated group. The data represent mean +_SE of 3 experiments.

94

the homogenate prepared from 1,6-BPq-fed larvae was stronger than that of the control larvae. In the homogenates, the effect of phenobarbital treatment appeared as a CL increase of the 1,6BPq-fed group. In the homogenate samples the CL intensity was 4 times stronger for the phenobarbital-treated group than for the non-treated group. Table 1 shows the mutation frequency by PAils in the Drosophila wing spot test. The frequency of small single spots induced by PAHs was higher than that in the control group. The frequency ratios against control (1.00) were 1.34, 1.34, 1.48, 1.82, 2.03 and 2.36 for anthracene, pyrene, 1,6-Pq, 1,8-Pq, 1,6-BPq and 3,6-BPq, respectively. 1,6-Pq, 1,8-Pq, 1,6-BPq and 3,6-BPq gave positive results in the statistical analysis of Drosophila mutagenicity tests according to the Kastenbaum-Bowman tables at the 5% level of significance (Frei and Wiirgler, 1988). The frequencies of large single spots and those of twin spots were not elevated above the control level. The realationship between the mutation frequency of small single spots in the Drosophila wing spot test and the CL from Drosophila melanogaster fed the PAHs is shown in Fig. 3. It was found that with various PAHs containing quinones such as 1,6-Pq, 1,8-Pq, 1,6-BPq and 3,6-BPq, there was a positive correlation between the mutation frequency in the Drosophila wing spot test and the CL intensity from Drosophila melanogaster fed those drugs. Anthracene was well known to be a non-carcinogenic PAH (Nesnow et al., 1986; Knuiman et al., 1987; Bos et

3.6-BPq =="i 0.4 c

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0

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o

looo

2000

~

3ooo

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40'oo

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Fig. 3. Mutation frequencies and chemiluminescence of P A l l quinones. The mutation frequencies were determined in the Drosophila wing spot test and the chemiluminescence intensities were measured on a homogenate of 50 pupae with 4 ml PBS.

Drosophila melanogaster fed

al., 1988). Pyrene was not mutagenic in Salmonella typhimurium TA100 without $9 mix and weakly mutagenic in Salmonella typhimurium TA100 with $9 mix (Furukawa et al., 1989). In this experiment, they gave negative results in the statistical analysis of Drosophila mutagenicity tests according to the Kastenbaum-Bowman tables at the 5% level of significance (Table 1) and their CL intensities were weaker than those of other mutagenic PAH quinones. In 1984, Hirono et al. identified a ptaquiloside as the most carcinogenic compound in bracken fern. Fig. 4 shows the CL from Drosophila melanogaster fed 10% or 34% bracken fern. The CL intensity from living intact larvae fed bracken fern increased 2.2 times with the 10% addition and 6.8 times with the 34% addition as compared to the controls which had not received bracken fern. The CL intensity from intact adults fed

TABLE 1 M U T A N T SPOT F R E Q U E N C Y P E R W I N G A N D D I A G N O S I S O F S M A L L SINGLE W I N G SPOTS I N D U C E D BY P O L Y C Y C L I C A R O M A T I C H Y D R O C A R B O N S IN T H E D R O S O P H I L A W I N G SPOT SYSTEM Fly

Control 1,6-Pq 1,8-Pq 1,6-BPq 3,6-BPq Pyrene Anthracene

Single spots small

large

Twin spots

N u m b e r of wings scored

Diagnosis of small single spots

0.33 0.49 0.60 0.67 0.78 0.44 0.44

0.05 0.02 0.05 0.08 0.03 0 0.07

0.02 0.02 0.02 0 0 0 0

104 104 100 92 58 51 51

positive positive positive positive negative negative

The statistical tests were carried out according to the K a s t e n b a u m - B o w m a n tables at the 5% level of significance.

95

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chemiluminesenee change -50

-40

i

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-30

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-20

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I

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& Yamada)L,5000 (Hirono 11984)

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control

10%

bracken

34%

control

fern-fed

7]

2315+-578tcpm)

10%

34%

bracken

fern-fed

Fig. 4. Chemiluminescence from Drosophila melanogaster fed bracken fern powder. The hatched bars indicate the living intact group. The white bars show the group homogenized with 4 ml PBS. The data represent the mean+++SE of 3 experiments.

bracken fern also increased. In the case of homogenates, the CL intensity from larvae fed 34% bracken fern powder was 1.5 times stronger than that of control preparations. There was the same tendency in adults. It was found that the CL difference between untreated flies and those dosed with bracken fern was clearer when living intact larvae were measured than with any other sample. Fig. 5 shows the PCOOH concentration in total lipid obtained from Drosophila melanogaster larvae fed bracken fern. With 10% bracken fern, the PCOOH concentration in total lipid increased 1.9 times. With 34% bracken fern, it increased significantly by 4.2 times.

~>

(4.2)

4o

~ 30

6

-o 20 E

(1.9)

"~ Q 10 O a.

0

control

10%

34%

bracken fern-fed

Fig. 5. Phosphatidylcholine hydroperoxide ( P C O O H ) in total lipid of Drosophila melanogaster. The total lipid was obtained from larvae fed with bracken fern. P C O O H was determined by CL-HPLC. Figures in parentheses show the ratio against the control group.

Fig. 6. Effects of free radical scavengers and active oxygen quenchers on the chemiluminescence of adult fly homogenate fed 10% bracken fern powder. The CL in the absence of additions was 2315 _+579 cpm (mean +_SE). Additions: GSH, glutathione (10 raM); SOD, superoxide dismutase (500 U / m l ) ; catalase (500 U / m l ) ; Tiron (5 mM); L-ASA, L-ascorbic acid (33 mM).

The effect of free radical scavengers on CL from adult fly homogenate fed 10% bracken fern powder is shown in Fig. 6. The CL intensity decreased in the presence of free radical scavengers and active oxygen quenchers such as catalase (H20 2 scavenger), glutathione (radical scavenger), superoxide dismutase (SOD, superoxide scavenger), Tiron (superoxide scavenger, and chelating agent inhibiting the Fenton reaction and blocking heme-protein enzymes) and Lascorbic acid (radical scavenger). In particular the addition of catalase inhibited 50% of the fly CL. Discussion

The spontaneous CL from Drosophila melanogaster fed some mutagens was detected for the first time by the use of a very sensitive synchronous single photon counting apparatus (Figs. 2 and 4). CL is generally observed when a free radical reaction is provoked and photo-emissive active oxygens (such as singlet oxygen) and electrically excited species (such as triplet carbonyls) are generated in the living tissues and bodies (Miyazawa and Kaneda, 1981; Boveris et al., 1981). It has been reported that some mwtagens like quinones exerted cytotoxic effects via oxygen reduction and redox cycling and the superoxide radical (Kappus and Sies, 1981). It has been reported that the microsomal CL observed by incub a t i o n with 7 , 8 - d i h y d r o x y - 7 , 8 - d i h y d r o benzo[ a]pyrene (7,8-diol-BP) is much greater than

96 with other PAHs which had lower carcinogenic activities (Hamman et al., 1981). The CL from Drosophila melanogaster fed P A H quinones showed a linear relation with the mutation frequency in the Drosophila wing spot test (Fig. 3). This suggests that free radical evolution is closely involved with mutagenic reactions and influences fly CL intensities. The effect of bracken fern administration on CL was much more definitive at the larval stage than at the adult stage (Fig. 4). In the larval stage, cell division seems to be most active. Somatic mutation does not occur without cell division (Fujikawa, 1988). The CL enhancement in living larvae by bracken fern administration was clearer than in the homogenate (Fig. 4). These CL results suggest that the bracken fern-dependent CL correlates closely with active cell division. The phenobarbital treatment did not have any effect on the CL of living larvae fed 1,6-BPq (Fig. 2). It has been reported that the benzo[a]pyrenedependent CL in mice was enhanced by phenobarbital treatment to induce the liver mixed-function oxidase (Yoshida et al., 1989). This may reflect non-enzymatic conversion of 1,6-BPq to the mutagenic compound. The CL intensity from Drosophila rnelanogaster fed bracken fern decreased by addition of radical scavengers and active oxygen quenchers (Fig. 6). This suggests that active oxygen plays a role in the CL from bracken fern-fed flies. Bracken fern was sensitive in the Ames assay with TA102, which acts on oxidative mutagens (Furukawa, personal communication). This suggests that Drosophila melanogaster fed bracken fern produce large amounts of free radicals in the process of mutation in the fly body. Phosphatidylcholine (PC) is one of the most important components of biological membranes and its peroxidation gives rise to P C O O H as a primary product (Miyazawa et al., 1988b). As shown in the present experiment, the P C O O H concentration in larval lipid actually increased by bracken fern administration (Fig. 5). It seems possible that lipid peroxidation was stimulated in the process of mutation in Drosophila melanogaster. The CL reaction observed in fly bodies seems to involve lipid peroxidation as a result of

free radical reactions. The breakdown of lipid hydroperoxide by heme proteins, such as cytochromes, generates singlet oxygen and excited triplet carbonyls, and those compounds are photo-emissive (give emission bands around 500800 nm) when they transit to their energetically lower states such as triplet oxygen and singlet carbonyls as previously observed in spectrophotometric studies on animal tissues (Miyazawa et al., 1988b). In conclusion, the CL results obtained in the present study suggest that free radical evolutions play a role in the process of mutation in

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97 Meeting of the Society for Free Radical Research, Kyoto, Japan, 9-13 April 1988, pp. 1503-1506. Graf, U., F.E. Wiirgler, A.J. Katz, H. Frei, H. Juon, C.B. Hall and P.G. Kale (1984) Somatic mutation and recombination test in Drosophila melanogaster, Environ. Mutagen., 6, 153-188. Hamman, J.P., and H.H. Seliger (1976) The chemical formation of excited states during hydroxylation of the carcinogenic hydrocarbon benzo[a]pyrene by liver microsomes, Biochem. Biophys. Res. Commun., 70, 675-680. Hamman, J.P., H.H. Seliger and G.H. Posner (1981) Specificity of chemiluminescence in the meatbolism of benzo[a]pyrene to its carcinogenic diol epoxide, Proc. Natl. Acad. Sci. (U.S.A.), 78, 940-942. Hirono, I., K. Yamada, H. Niwa, Y. Shizuri, M. Ojika, S. Hosaka, T. Yamaji, K. Wakamatsu, H. Kigoshi, K. Niiyama and Y. Uosaki (1984) Separation of carcinogenic fraction of bracken fern, Cancer Lett., 21,239-246. Inaba, H., A. Yamagishi, C. Takyu, B. Yoda, Y. Goto, T. Miyazawa, T. Kaneda and A. Saeki (1982) Development of an ultra-high sensitive photon counting system and its application to biomedical measurements, Optics Lasers Eng., 3, 125-130. Kappus, H., and H. Sies (1981) Toxic drug effects associated with oxygen metabolism: redox cycling and lipid peroxidation, Experientia, 37, 1233-1358. Knuiman, M.W., N.M. Laird and T.A. Louis (1987) Interlaboratory variability in Ames assay results, Mutation Res., 180, 171-182. Miyazawa, T. (1989) Determination of phospholipid hydroperoxides in human blood plasma by a chemiluminescenceHPLC assay, Free Radical Biol. Med., 7, 209-217. Miyazawa, T., and T. Kaneda (1981) Extra-weak chemiluminescence of organ homogenate and blood in tocopheroldeficient rats, J. Nutr. Sci. Vitaminol., 27, 415-423.

Miyazawa, T., K. Yasuda, K. Fujimoto and T. Kaneda (1988a) Determination of phosphatidylcholine hydroperoxide in human plasma by chemiluminescence-high performance liquid chromatography, Anal. Lett., 21, 1033-1044. Miyazawa, T., F. Fujimoto and T. Kaneda (1988b) Lipid peroxidation and chemiluminescence, in: A. Sevanian (Ed.), Lipid Peroxidation in Biological Systems, AOCS Monograph, Am. Oil Chem. Soc., Champaign, IL, pp. 1-17. Nesnow, S., M. Argus, H. Bergman, K. Chu, C. Frith, T. Helmes, R. McGaughy, V. Ray, T.J. Slaga, R. Tennant and E. Weisburger (1986) Chemical carcinogens, a review and analysis of the literature of selected chemicals and the establishment of the Gene-Tox Carcinogen Data Base, A Report of the U.S. Environmental Protection Agency Gene-Tox Program, Mutation Res., 185, 1-195. Roberts, D.B. (1986) Basic Drosophila care and techniques. 6. Food, in: D.B. Roberts (Ed.), Drosophila, A Practical Approach, IRL Press, Oxford, pp. 15-17. Seliger, H.H., A. Thompson, J.P. Hamman and G.H. Posner (1982) Chemiluminescence of benzo[a]pyrene-7,8-diol, Photochem. Photobiol., 36, 359-365. Vassil'ev, R.F., and A.A. Vichutinskii (1962) Chemiluminescence and oxidation, Nature (London), 194, 1276-1277. Yoo, M.A., H. Ryo, T. Todo and S. Kondo (1985) Mutagenic potency of heterocyclic amines in the Drosophila wing spot test and its correlation to carcinogenic potency, Jap. J. Cancer Res. (Gann), 76, 468-473. Yoshida, LS., T. Miyazawa, K. Fujimoto and T. Kaneda (1989) Spontaneous and luminol-dependent chemiluminescences from tissue preparations of benzo[a]pyrene-injected mice, J. Nutr. Sci. Vitaminol., 35, 569-578.