Mutation Research, 237 (1990) 165-171 Elsevier
165
MUTAGI 09056
A m e t h y l viologen-sensitive m u t a n t of the n e m a t o d e Caenorhabditis elegans Naoaki Ishii 1, Kiyoko Takahashi 1, Satoru Tomita 2, Tetsuo Keino 3, Shuji Honda 4, Kazuhiro Yoshino 5 and Kenshi Suzuki 1 t Department of Molecular Biology, School of Medicine, Tokai University, Isehara, Kanagawa 259-11, 2 Radioisotope Laboratory, Fujigaoka Hospital, Showa University, Fujigaoka, Midoriku, Yokohama City 227, 3 Tokyo Institute, Celci Cosmetics Co., Matsue, Edogawa-ku, Tokyo 132, 4 Isotope Laboratory, Tokyo Metropolitan Institute of Gerontology, Sakae, Itabashi-ku, Tokyo 173 and 5 Institute for Control of Agin~ Haruoka, Fukuroi, Shizuoka 437-01 (Japan) (Received 2 April 1990) (Revision received 30 June 1990) (Accepted 9 July 1990)
Keywords: Caenorhabditis elegans; Paraquat-sensitive mutant; Oxygen toxicity; Life span
Summary A methyl viologen (paraquat)-sensitive mutant, mev-1 (LG III), in Caenorhabditis elegans was about 4 times more sensitive to methyl viologen than the wild type. This mutant was also hypersensitive to oxygen. The brood size was about 1 / 4 that of the wild type. The average life span was determined to be 9.3 days as compared to 14.3 days for the wild type. The activity of superoxide dismutase (SOD), a scavenging enzyme for superoxide anion, was about half the wild-type level. We suggest that oxygen radicals may be involved in the normal aging mechanism in C. elegans.
It has been proposed that free radicals, especially those of molecular oxygen, may accelerate aging in animals (Harman, 1968). Cutler (1984) has provided a variety of data which support this view. In particular, the superoxide dismutase (SOD) activity (E.C. 1.15.1.1) of several organs, a scavenger of oxygen free radicals (Fridovich, 1974), is positively correlated with the maximum life span for various animal species including primates. Similar correlations were observed for several other radical scavengers including plasma urate, carotinoids, and vitamin E. Interestingly, the product of the standard metabolic rate and the maximum
Correspondence: Dr. Naoaki Ishii, Department of Molecular Biology, School of Medicine, Tokai University, Isehara, Kanagawa 259-11 (Japan).
life span, called the LEP, is roughly constant for various animals, indicating that animals with lower standard metabolic rates can live for longer periods than animals with higher rates. To investigate the possible role of oxygen free radicals in aging, mutants of the nematode C. elegans were isolated which are hypersensitive to methyl viologen (paraquat). The toxic effects of this herbicidal drug on cells and animals are believed to be mediated by superoxide anions (Bagley et al., 1986; Krall et al., 1988). Here we report the properties of a methyl oiologen-sensitive mutant called mev-1. These mutants have only about half the wild-type level of SOD activity and are hypersensitive to gaseous oxygen as well as methyl viologen. They also have smaller brood sizes and a shorter average life span than the wild type. We suggest that superoxide anions are at
0921-8734/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
166
least partially responsible for the shortened life span of mev-I mutants and perhaps for normal aging in C. elegans.
viologen (0 0.2 mM). Four days later, the numbers of animals surviving to late larvae or adults were determined.
Materials and methods
Fecundity and life span Late-stage larvae were cultured individually on seeded, N G agar plates (15 mm) and then transferred to fresh plates daily as adults to avoid overgrowth of the plates by their progeny. Eggs deposited on these plates were counted after removing the adult. The life spans of individual adult hermaphrodites were similarly determined. The animals were examined daily to determine the day of death.
Nematode strains and general procedures Wild-type (designated N2) and mutant strains of C. elegans var. Bristol were obtained from the Caenorhabditis Genetics Center. Stock maintenance and handling were as described by Brenner (1974). In particular, nematodes were grown at 2 0 ° C on N G agar plates and live bacteria (Escherichia coli strain OP50, a uracil-requiring mutant) were added as food (Brenner, 1974). Ethyl methanesulfonate (EMS) and methyl viologen were obtained from Sigma Chemical Co. Isolation of mutants Methyl viologen-sensitive mutants were isolated after EMS mutagenesis (Brenner, 1974). Two screening steps were employed. In the initial screening, the F2 progeny of mutagenized hermaphrodites were treated as gravid adults with 30 mM methyl viologen in M9 buffer for 4 h at 20 ° C. After this treatment, inactive animals were individually cultured on small, seeded N G agar plates. Whereas wild-type animals survive this treatment, methyl viologen-sensitive animals may die. Because methyl viologen does not penetrate eggshells, viable embryos can still be recovered from the treated adults after transfer to drug-free plates. For the rescreening, asynchronous cultures of progeny from each initial isolate were grown on N G agar plates. Eggs were collected from these cultures using sodium hypochlorite (Emmons et al., 1979) and hatched by overnight incubation at 2 0 ° C in S buffer (Sulston and Brenner, 1974). The newly hatched L 1 larvae were cultured on seeded N G agar plates containing 0.2 m M methyl viologen (200-400 worms per 5-cm plate). Three days later, the numbers of animals surviving to maturity were determined for each plate. From this rescreening, we obtained 2 independent strains in which the growth of larvae was strongly inhibited by methyl vioiogen. Sensitivity to methyl viologen L~ larvae were cultured on seeded, N G agar plates containing various concentrations of methyl
Sensitivity to oxygen gas Thirty L~ larvae were placed together on seeded, N G agar plates (15 mm) and then exposed to various concentrations of oxygen gas in an airtight plastic chamber. The gas in the chambers was replaced once a day and the oxygen concentration was monitored by an oxygen analyzer (Iijima Products Manufacturing Co. Model G-101-Y). The numbers of surviving animals were determined after 3 days of exposure. Superoxide dismutase activity Various assays have been devised for SOD activity and each has its characteristic advantage. All of these methods comprise a system for producing superoxide anions and a color reaction that monitors their consumption. In the present study, a xanthine/xanthine oxidase system (Misra and Fridovich, 1972) was used to produce superoxide anions while a h y d r o x y l a m i n e / N - l - n a p h t h y l e t h y lene diamine/sulfanilic acid system was used for detection. The residual superoxide anion combines with the hydroxylamine to produce nitrite. The nitrate, in turn, reacts with the latter 2 compounds to create a dye with an extinction maximum at 550 nm. The following solutions were added successively and incubated at 3 7 ° C for 30 min: K H 2 P O 4 (65 m M ) - b o r a t e (35 mM) buffer, p H 8.2, plus 0.5 mM EDTA, 0.2 ml; xanthine (0.5 mM), 0.2 ml: hydroxylamine HCI (10 mM) plus hydroxylamine- O-sulfonic acid (1 m g / m l ) , 0.1 ml; H 2 0 or K C N (5 mM), 0.2 ml; sample, 0.2 ml; xanthine oxidase (sigma), 4.8 × 10 -3 u n i t / m l , 0.2 ml. After
167
incubation, 2.0 ml of the coloring reagent (30 #M N-l-naphthylethylene diamine hydrochloride, 3 mM sulfanilic acid and 25% acetic acid) was added. The absorbance at 550 nm was measured in a Hitachi spectrophotometer (model 101) after one more hour incubation at room temperature. This method has been developed and described by Oyanagi (1984). For enzyme measurements, nematodes were grown at 2 0 ° C with continuous shaking in a liquid medium prepared by adding 12 g Yeast Extract (Difco) and 12 g Bacto Soytone (Difco) to 360 ml distilled water. After autoclaving and cooling, 10 ml penicillin G (4000 units/ml), 10 ml streptomycin sulfate (8 mg/ml), 40 ml myoglobin (5 mg/ml), and 4 ml steroid mixture (prepared by dissolving 60 mg fl-sitosterol, 20 mg cholesterol, 20 mg ergosterol and 2.5 ml Tween 80 into 17.5 ml distilled water) were added (Rothstein, 1974). Each of these additives was first sterilized by filtration. After 2 weeks the number of animals reached about 1.0 × 105/ml. These were then harvested by centrifugation, washed, and stored frozen in liquid nitrogen. The frozen animals were powdered in a mortar cooled with dry ice, and extracted in S buffer. Protein concentrations were determined using the BioRad Protein Assay Reagent with bovine serum albumin as standard. Results
Mutation isolation and mapping About 15,000 F2 progeny of EMS-mutagenized hermaphrodites were examined for hypersensitivity to methyl viologen. During the initial screening, about 360 candidates were isolated, which presumably included many individuals that died of reasons unrelated to the drug treatment. In the rescreening, 2 strains were proved to be highly sensitive to methyl viologen. One strain, mev-1, was back-crossed 5 times with wild-type males using methyl viologen sensitivity as a marker to identify mutant progeny. It was found that the mev-1 ( k n l ) mutation behaves as a single, recessive Mendelian factor on linkage group III. It was positioned by 3-factor crosses: dpy-17 (8/8) (unc32 mev-1); unc-32 (3/13) mev-1 (10/13) dpy-18; mev-1 dpy-18 (13/13) unc-64. In another strain
CONCENTRATION 0 100
0.05
OF M E V (raM)
0.10 j
0.15 I
0.20
z O m Io er u,. 10 r,..3 z n,.
1 O
Fig. 1. Sensitivity of mev-! mutants to methyl viologen. L l larvae were cultured on N G agar containing methyl viologen at various concentrations and their survival was scored after 4 days. Vertical bars represent standard deviations from 4 replicate experiments. Approximately 100 animals were scored for each strain. Closed circles, wild type; open circles, mev-l.
the mutation was located on linkage group X (unpublished).
Sensitivity to methyl viologen L 1 larvae were cultured on plates containing various concentrations of methyl viologen. At the highest concentration of methyl viologen examined (0.2 mM), most wild-type animals develop into L 4 larvae or adults within 4 days. However, mev-1 mutants usually arrested as L a or L 2 larvae and were easily distinguished from the wild type. This technique was first developed by Hartman and Herman (1982) to isolate radiation-sensitive mutants of C. elegans. At 0.2 mM methyl viologen, some 60% of wildtype animals survived, while less than 1% of mev-1
168
15(
Joo~
m
> >
t~
: i
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~100
Z Ot)
wild
(3
(3 LU
" . . . . .
i0
type
mev-1
U.
O
n- 50
DAYS
15
20
7
AFTER HATCH
Fig. 3. Life span of mev-I mutants. Approximately 100 animals were observed for each strain. One of 2 experiments is shown because the results were similar. Closed circles, wild type; open circles, mev-1.
UJ
m Z
begins to decline earlier in mev-1. Thus the average life span of mev-1 mutants is only 9.4 days compared to 14.3 days for the wild type.
,
O
J
'1-
2
4
6
DAYS A F T E R
8
10
OXYGEN
12 0
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_
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~
C O N C E N T R A T I O N (%)
40
6• ;
80 100 - , "--,
Fig. 2. The number of eggs laid by mev-I and wild-type hermaphrodites. Ten animals were observed for the wild type and 15 for rnev-1. Vertical lines represent standard deviations.
mutants survived (Fig. 1). Comparing the concentrations causing 40% lethality, the mev-1 mutants are about 4 times more sensitive than the wild type.
z
9 o iJ.
Fecundity The mev-1 mutants have normal morphology. However, they have low vital activity, i.e., slow growth and low fecundity. Egg-laying in the wild type begins on the third day after hatching, reaches a m a x i m u m of about 130 eggs per animal per day on the fifth day and continues for another week (Fig. 2). The average number of eggs laid per individual was 287 + 34 (n = 10). The onset of egg production was delayed for about half a day in mev-1 mutants and the average number of eggs laid per animal was only 77 + 48 (n = 15). Life span A typical survival curve is shown in Fig. 3. The m a x i m u m life span (21 days) is nearly the same for rnev-1 mutants and the wild type but survival
10 z
!
Fig. 4. Sensitivity of mev-1 mutants to oxygen gas. L~ larvae were exposed to oxygen/nitrogen mixtures at various concentrations and their survival was scored after 3 days. Vertical bars represent standard deviations from 4 replicate experiments. Approximately 100 animals were scored for each strain. Closed circles, wild type; open circles, mev-1.
169 TABLE 1 SOD ACTIVITY IN T H E mev-I A N D WILD TYPE OF C.
elegans Strain
Activity a (units)
Mn-SOD b (%)
Wild type
77.8 90.0 39.2 41.6
almost 0 almost 0 14 21
mev-1
a Data from 2 experiments are shown as units per mg protein. b Percent activity insensitive to 1 mM KCN of total activity.
Sensitivity to oxygen gas Interestingly, mev-1 mutants are even more hypersensitive to oxygen gas than to methyl viologen (Fig. 4). Whereas wild-type animals can develop nearly normally under 90% oxygen, few mev-1 larvae survive a 3-day exposure. The growth and movement of mev-1 larvae were nearly normal for the first day of exposure, but arrested thereafter. SOD activity Superoxide dismutase in eukaryotic cells is composed of 2 types, one containing Zn and Cu in the active center and another containing Mn (Halliwell and Cutteridge, 1985). The activities of these 2 types can be distinguished by adding 1 m M KCN to the reaction mixtures, which specifically inhibits the Z n / C u enzyme. The SOD activity in mev-1 is about 50% of that in the wild type (Table 1). Most wild-type activity is inhibited by KCN, indicating that there is relatively little, if any, Mn-SOD. In contrast, 14-21% of the residual activity in mev-1 could be attributed to the Mn-SOD. As a caveat, these measurements were made from asynchronous populations and are conceivably affected by unrecognized differences in the stage distributions of the populations. Discussion
A C. elegans mutant was isolated which is about 4 times more sensitive to methyl viologen (paraquat) than the wild type. The gene was designated mev-1. Genetic analysis revealed that the mev-1 mutation is located on linkage group III.
These mutants have low fecundity and consequently populations grow slowly. As there are other C. elegans mutants with small brood sizes, this is not a specific property of mev-1 (Swanson et al., 1984). The mev-1 mutants were found to be hypersensitive to oxygen gas as well as methyl viologen and have reduced Z n / C u - S O D activity. This suggests that the toxicity of methyl viologen is mediated, at least in part, by its ability to generate superoxide anions (Bagley et al., 1986; Krall et al., 1988) and that the resistance of wildtype nematodes to methyl viologen is mediated by SOD (Hassan and Fridovich, 1978). Methyl viologen-sensitive or oxygen-sensitive mutants have been isolated in various organisms, including E. coli (Hassan and Fridovich, 1979; Fridovich, 1986; Carlioz and Touati, 1986), B. coagulans (Vassilyadi and Archibald, 1985), yeast (Bilinski et al., 1985; Van Loon et al., 1986) and Drosophila melanogaster (Tang et al., 1986; Phillips et al., 1989). In several cases, these mutants were defective in SOD (Hassan and Fridovich, 1979; Fridovich, 1986; Carlioz and Touati, 1986; Bilinski et al., 1985; Van Loon et al., 1986), but oxygen hypersensitivity need not be accompanied by reduced SOD activity (Carlioz and Touati, 1986). With possible rare exceptions, all known oxygen-tolerant organisms have SOD (Fridovich, 1983), suggesting that SOD could provide some oxygen tolerance to most organisms. SOD-defective mutants have been isolated recently in D. melanogaster (Tang et al., 1986; Phillips et al., 1989). One type of mutant (Tang et al., 1986) was found to be hypersensitive to X-rays and another type (Phillips et al., 1989) was completely defective in Cu/Zn-SOD, had a very short life span and was sensitive to methyl viologen. But sensitivity to oxygen was not examined for either type of mutant. Our mev-1 data are possibly the first mutant studies to suggest a correlation between oxygen or methyl viologen sensitivity and SOD activity in animals. In humans, 21-monosomy reduces SOD activity by about half compared to normal controls (Yoshimitsu et al., 1983; Nakai et al., 1984). In contrast, 21-trisomy (Down's syndrome) may result in overexpression of the SOD gene (Groner et al., 1986; but see Miyazaki et al., 1987; Jeziorowska et al., 1987). It has been pro-
170
posed that SOD overexpression may have an etiological role in Down's syndrome. Like the cSOD "1°8 mutant of Drosophila (Phillips et al., 1989), a characteristic of mev-1 strains is a shortened life span. The average life span of mev-1 animals is only 66% of the wild-type life span. The life span of the wild type remained constant over the range of 2-40% oxygen, while the life span of the mutant varied in dependence on the concentrations of oxygen (unpublished). We suggest that the reduced life span is caused by oxygen damage, and in particular by superoxide anions. The reduced life span could reflect an acceleration of normal aging processes. In any case, SOD appears to be required for a normal life span in C. elegans. According to Cutler (1984), the concentration of various endogenous antioxidants such as SOD, urate, or vitamin E, is positively correlated with the longevity of mammals, including primates. For SOD, however, the activity divided by the standard metabolic rate, rather than the SOD activity itself, is more linearly related to the maximum life span (Tolmasoff et al., 1984). Since we have not examined the standard metabolic rate of mev-1 or wild-type nematodes, comparison at this level is not yet possible. There is a recent report by Morimyo (1988) which deals with a methyl viologen-sensitive mutant of E. coli (mvr). However, the cloned gene did not hybridize with any of the cloned SOD genes (personal communication). Regardless of the structure and function of the mev-1 gene, this is the first report which deals with defective SOD activity in C. elegans.
Acknowledgements We are indebted to Dr. E.M. Hedgecock, Department of Biology, Johns Hopkins University and Dr. T. Johnson, University of Colorado, for their kind advice and encouragement during this study and for reading the manuscript. References Bagley, A.C., J. Krall and R.E. Lynch (1986) Superoxide mediates the toxicity of paraquat for Chinese hamster ovary cells, Proc. Natl. Acad. Sci. (U.S.A.), 83, 3189-3193.
Bilinski, T., Z. Krawiec, A. Liczmanski and J. Litwinska (1985) Is hydroxyl radical generated by the fenton reaction in vivo?, Biochem. Biophys. Res. C o m m u n . , 130, 533-539. Brenner, S. 1974) The genetics of Caenorhabditis elegans, Genetics, 77, 71-94. Carlioz, A., and A. Touati (1986) Isolation of superoxide dismutase m u t a n t s in Escherichia coli: is superoxide dismutase necessary for aerobic life?, EMBO J., 5, 623-630. Cutler, R.G. (1984) in: A.K. Roy and B. Chaterjee (Eds.), Molecular Basis of Aging, Academic Press, New York, NY. pp. 263-352. Emmons, S.W., M.R. Klass and D. Hirsch (1979) Analysis of the constancy of D N A sequences during development and evolution of the nematode Caenorhabditis elegans, Proc. Natl. Acad. Sci. (U.S.A.), 76, 1333-1337. Fridovich. 1. (1974) Superoxide dismutase, Adv. Enzymol., 4l, 35 -97. Fridovich, I. (1983) Superoxide radical: an endogenous toxicant, Annu. Rev. Pharmacol. Toxicol.. 239-257. Fridovich, 1. (1986) Biological effects of the superoxide radical, Arch. Biochem. Biophys., 247, 1 11. Groner, Y., O. Elroy-Steim Y. Bernstein, N. Dafni, D. LevAnon, E. Danciger and A. Neer (1986) Molecular genetics of Down's syndrome: overexpression of transfected h u m a n C u / Z n - s u p e r o x i d e dismutase gene and the consequent physiological changes, Cold Spring Harbor Syrup. Quant. Biol., 51, 381-393. Halliwell, N., and J.M.C. Gutteridge (1985) in: Free Radicals in Biology and Medicine, Clarendon. Oxford, pp. 84 100. Harman, D. (1968) Free radical theory of aging: effect of free radical reaction inhibitors on the mortality rate of male LAF mice, J. Geront., 23, 476-482. Hartman, P.S., and R.K. Herman (1982) Radiation-sensitive mutants of Caenorhabditis elegans, Genetics, 10, 159-178. Hassan, H.M., and I. Fridovich (1978) Superoxide radical and the oxygen enhancement of the toxicity of paraquat in Escherichia coli, J. Biol. Chem., 253, 8143-8148. Hassan, H.M., and I. Fridovich (1979) Superoxide, hydrogen peroxide, and oxygen tolerance of oxygen-sensitive mutants of Escherichia coli, Rev. Inf. Dis., 1, 357-367. Jeziorowska, A., L. Jakubowski, J. Lach and B. Kaluzewski (1987) Regular trisomy 21 not accompanied by increased copper-zinc superoxide dismutase (SOD1) activity, Clin. Genet., 33, 11-19. Krall, J., A.C. Bagley, G.T. Mullenbach, R.A. Hallewell and E. Lynch (1988) Paraquat-resistant HeLa cells, Basic Life Sci., 49, 811 814. Misra, H.P., and i. Fridovich (1972) The univalent reduction of oxygen by reduced flavins and quinones, J. Biol. Chem., 247, 188 192. Miyazaki, K., T. Yamanaka and N. Ogasawara (1987) A boy with Down's syndrome having recombinant chromosome 21 but no SOD-1 excess, Clin. Genet., 32, 383-387. Morimyo, M. (1988) Isolation and characterization of methyl viologen-sensitive m u t a n t s of Escherichia coli KI2, J. Bacteriol., 170, 2136-2142. Nakai, H., K. Tada and Y. Abe (1984) Erythrocyte superoxide dismutase-1 and 21 monosomy, Cytogenet. Cell Genet., 37, 547.
171 Oyanagi, Y. (1984) Establishment of nitrite-kit for SOD activity determination, Enshoh (Inflammation), 4, 63-73. Peng, T.X., A. Moya and F.J. Ayala (1986) Irradiation-resistance conferred by superoxide dismutase: possible adaptive role of a natural polymorphism in Drosophila melanogaster, Proc. Natl. Acad. Sci. (U.S.A.), 83, 684-687. Phillips, J.P., S.D. Campbell, D. Michaud, M. Charbonneau and A.J. Hilliker (1989) Null mutations of copper/zinc superoxide dismutase in Drosophila confer hypersensitivity, Proc. Natl. Acad. Sci. (U.S.A.), 86, 2761-2765. Rothstein, M. (1974) Practical methods for the axenic culture of the free-living nematodes Turbatrix aceti and Caenorhabditis briggsae, Comm. Biochem. Physiol., 49B, 669-678. Sulston, J.E., and S. Brenner (1974) The DNA of Caenorhabditis elegans, Genetics, 77, 95-104. Swanson, M.M., M.L. Edgley and D.L. Riddle (1984) in: S.J. O'Brien (Ed.), Genetic Maps, Vol. 3, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 286-299.
Tolmasoff, J.M., T. Ono and R,G. Cutler (1984) Superoxide dismutase: correlation with life-span and specific metabolic rate in primate species, Proc. Natl. Acad. Sci. (U.S.A.), 77, 2777-2781. Van Loon, A.P.G.M., B. Pesold-Hurt and G. Schatz (1986) A yeast mutant lacking mitochondrial manganese-superoxide dismutase is hypersensitive to oxygen, Proc. Natl. Acad. Sci. (U.S.A.), 83, 3820-3824. Vassilyadi, M., and F. Archibald (1985) Catalase, superoxide dismutase, and the production of O2-sensitive mutants of Bacillus coagulans, Can. J. Microbiol., 31, 994-999. Yoshimitsu, K., S. Hatano, Y. Kobayashi, Y. Takeoka, M. Hayashidani, K. Ueda, K. Nomura, K. Ohama and T. Usui (1983) A case of 21q-syndrome with half normal SOD-1 activity, Hum. Genet., 64, 200-202.
165
MUTAGI 09056
A m e t h y l viologen-sensitive m u t a n t of the n e m a t o d e Caenorhabditis elegans Naoaki Ishii 1, Kiyoko Takahashi 1, Satoru Tomita 2, Tetsuo Keino 3, Shuji Honda 4, Kazuhiro Yoshino 5 and Kenshi Suzuki 1 t Department of Molecular Biology, School of Medicine, Tokai University, Isehara, Kanagawa 259-11, 2 Radioisotope Laboratory, Fujigaoka Hospital, Showa University, Fujigaoka, Midoriku, Yokohama City 227, 3 Tokyo Institute, Celci Cosmetics Co., Matsue, Edogawa-ku, Tokyo 132, 4 Isotope Laboratory, Tokyo Metropolitan Institute of Gerontology, Sakae, Itabashi-ku, Tokyo 173 and 5 Institute for Control of Agin~ Haruoka, Fukuroi, Shizuoka 437-01 (Japan) (Received 2 April 1990) (Revision received 30 June 1990) (Accepted 9 July 1990)
Keywords: Caenorhabditis elegans; Paraquat-sensitive mutant; Oxygen toxicity; Life span
Summary A methyl viologen (paraquat)-sensitive mutant, mev-1 (LG III), in Caenorhabditis elegans was about 4 times more sensitive to methyl viologen than the wild type. This mutant was also hypersensitive to oxygen. The brood size was about 1 / 4 that of the wild type. The average life span was determined to be 9.3 days as compared to 14.3 days for the wild type. The activity of superoxide dismutase (SOD), a scavenging enzyme for superoxide anion, was about half the wild-type level. We suggest that oxygen radicals may be involved in the normal aging mechanism in C. elegans.
It has been proposed that free radicals, especially those of molecular oxygen, may accelerate aging in animals (Harman, 1968). Cutler (1984) has provided a variety of data which support this view. In particular, the superoxide dismutase (SOD) activity (E.C. 1.15.1.1) of several organs, a scavenger of oxygen free radicals (Fridovich, 1974), is positively correlated with the maximum life span for various animal species including primates. Similar correlations were observed for several other radical scavengers including plasma urate, carotinoids, and vitamin E. Interestingly, the product of the standard metabolic rate and the maximum
Correspondence: Dr. Naoaki Ishii, Department of Molecular Biology, School of Medicine, Tokai University, Isehara, Kanagawa 259-11 (Japan).
life span, called the LEP, is roughly constant for various animals, indicating that animals with lower standard metabolic rates can live for longer periods than animals with higher rates. To investigate the possible role of oxygen free radicals in aging, mutants of the nematode C. elegans were isolated which are hypersensitive to methyl viologen (paraquat). The toxic effects of this herbicidal drug on cells and animals are believed to be mediated by superoxide anions (Bagley et al., 1986; Krall et al., 1988). Here we report the properties of a methyl oiologen-sensitive mutant called mev-1. These mutants have only about half the wild-type level of SOD activity and are hypersensitive to gaseous oxygen as well as methyl viologen. They also have smaller brood sizes and a shorter average life span than the wild type. We suggest that superoxide anions are at
0921-8734/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
166
least partially responsible for the shortened life span of mev-I mutants and perhaps for normal aging in C. elegans.
viologen (0 0.2 mM). Four days later, the numbers of animals surviving to late larvae or adults were determined.
Materials and methods
Fecundity and life span Late-stage larvae were cultured individually on seeded, N G agar plates (15 mm) and then transferred to fresh plates daily as adults to avoid overgrowth of the plates by their progeny. Eggs deposited on these plates were counted after removing the adult. The life spans of individual adult hermaphrodites were similarly determined. The animals were examined daily to determine the day of death.
Nematode strains and general procedures Wild-type (designated N2) and mutant strains of C. elegans var. Bristol were obtained from the Caenorhabditis Genetics Center. Stock maintenance and handling were as described by Brenner (1974). In particular, nematodes were grown at 2 0 ° C on N G agar plates and live bacteria (Escherichia coli strain OP50, a uracil-requiring mutant) were added as food (Brenner, 1974). Ethyl methanesulfonate (EMS) and methyl viologen were obtained from Sigma Chemical Co. Isolation of mutants Methyl viologen-sensitive mutants were isolated after EMS mutagenesis (Brenner, 1974). Two screening steps were employed. In the initial screening, the F2 progeny of mutagenized hermaphrodites were treated as gravid adults with 30 mM methyl viologen in M9 buffer for 4 h at 20 ° C. After this treatment, inactive animals were individually cultured on small, seeded N G agar plates. Whereas wild-type animals survive this treatment, methyl viologen-sensitive animals may die. Because methyl viologen does not penetrate eggshells, viable embryos can still be recovered from the treated adults after transfer to drug-free plates. For the rescreening, asynchronous cultures of progeny from each initial isolate were grown on N G agar plates. Eggs were collected from these cultures using sodium hypochlorite (Emmons et al., 1979) and hatched by overnight incubation at 2 0 ° C in S buffer (Sulston and Brenner, 1974). The newly hatched L 1 larvae were cultured on seeded N G agar plates containing 0.2 m M methyl viologen (200-400 worms per 5-cm plate). Three days later, the numbers of animals surviving to maturity were determined for each plate. From this rescreening, we obtained 2 independent strains in which the growth of larvae was strongly inhibited by methyl vioiogen. Sensitivity to methyl viologen L~ larvae were cultured on seeded, N G agar plates containing various concentrations of methyl
Sensitivity to oxygen gas Thirty L~ larvae were placed together on seeded, N G agar plates (15 mm) and then exposed to various concentrations of oxygen gas in an airtight plastic chamber. The gas in the chambers was replaced once a day and the oxygen concentration was monitored by an oxygen analyzer (Iijima Products Manufacturing Co. Model G-101-Y). The numbers of surviving animals were determined after 3 days of exposure. Superoxide dismutase activity Various assays have been devised for SOD activity and each has its characteristic advantage. All of these methods comprise a system for producing superoxide anions and a color reaction that monitors their consumption. In the present study, a xanthine/xanthine oxidase system (Misra and Fridovich, 1972) was used to produce superoxide anions while a h y d r o x y l a m i n e / N - l - n a p h t h y l e t h y lene diamine/sulfanilic acid system was used for detection. The residual superoxide anion combines with the hydroxylamine to produce nitrite. The nitrate, in turn, reacts with the latter 2 compounds to create a dye with an extinction maximum at 550 nm. The following solutions were added successively and incubated at 3 7 ° C for 30 min: K H 2 P O 4 (65 m M ) - b o r a t e (35 mM) buffer, p H 8.2, plus 0.5 mM EDTA, 0.2 ml; xanthine (0.5 mM), 0.2 ml: hydroxylamine HCI (10 mM) plus hydroxylamine- O-sulfonic acid (1 m g / m l ) , 0.1 ml; H 2 0 or K C N (5 mM), 0.2 ml; sample, 0.2 ml; xanthine oxidase (sigma), 4.8 × 10 -3 u n i t / m l , 0.2 ml. After
167
incubation, 2.0 ml of the coloring reagent (30 #M N-l-naphthylethylene diamine hydrochloride, 3 mM sulfanilic acid and 25% acetic acid) was added. The absorbance at 550 nm was measured in a Hitachi spectrophotometer (model 101) after one more hour incubation at room temperature. This method has been developed and described by Oyanagi (1984). For enzyme measurements, nematodes were grown at 2 0 ° C with continuous shaking in a liquid medium prepared by adding 12 g Yeast Extract (Difco) and 12 g Bacto Soytone (Difco) to 360 ml distilled water. After autoclaving and cooling, 10 ml penicillin G (4000 units/ml), 10 ml streptomycin sulfate (8 mg/ml), 40 ml myoglobin (5 mg/ml), and 4 ml steroid mixture (prepared by dissolving 60 mg fl-sitosterol, 20 mg cholesterol, 20 mg ergosterol and 2.5 ml Tween 80 into 17.5 ml distilled water) were added (Rothstein, 1974). Each of these additives was first sterilized by filtration. After 2 weeks the number of animals reached about 1.0 × 105/ml. These were then harvested by centrifugation, washed, and stored frozen in liquid nitrogen. The frozen animals were powdered in a mortar cooled with dry ice, and extracted in S buffer. Protein concentrations were determined using the BioRad Protein Assay Reagent with bovine serum albumin as standard. Results
Mutation isolation and mapping About 15,000 F2 progeny of EMS-mutagenized hermaphrodites were examined for hypersensitivity to methyl viologen. During the initial screening, about 360 candidates were isolated, which presumably included many individuals that died of reasons unrelated to the drug treatment. In the rescreening, 2 strains were proved to be highly sensitive to methyl viologen. One strain, mev-1, was back-crossed 5 times with wild-type males using methyl viologen sensitivity as a marker to identify mutant progeny. It was found that the mev-1 ( k n l ) mutation behaves as a single, recessive Mendelian factor on linkage group III. It was positioned by 3-factor crosses: dpy-17 (8/8) (unc32 mev-1); unc-32 (3/13) mev-1 (10/13) dpy-18; mev-1 dpy-18 (13/13) unc-64. In another strain
CONCENTRATION 0 100
0.05
OF M E V (raM)
0.10 j
0.15 I
0.20
z O m Io er u,. 10 r,..3 z n,.
1 O
Fig. 1. Sensitivity of mev-! mutants to methyl viologen. L l larvae were cultured on N G agar containing methyl viologen at various concentrations and their survival was scored after 4 days. Vertical bars represent standard deviations from 4 replicate experiments. Approximately 100 animals were scored for each strain. Closed circles, wild type; open circles, mev-l.
the mutation was located on linkage group X (unpublished).
Sensitivity to methyl viologen L 1 larvae were cultured on plates containing various concentrations of methyl viologen. At the highest concentration of methyl viologen examined (0.2 mM), most wild-type animals develop into L 4 larvae or adults within 4 days. However, mev-1 mutants usually arrested as L a or L 2 larvae and were easily distinguished from the wild type. This technique was first developed by Hartman and Herman (1982) to isolate radiation-sensitive mutants of C. elegans. At 0.2 mM methyl viologen, some 60% of wildtype animals survived, while less than 1% of mev-1
168
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m
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: i
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wild
(3
(3 LU
" . . . . .
i0
type
mev-1
U.
O
n- 50
DAYS
15
20
7
AFTER HATCH
Fig. 3. Life span of mev-I mutants. Approximately 100 animals were observed for each strain. One of 2 experiments is shown because the results were similar. Closed circles, wild type; open circles, mev-1.
UJ
m Z
begins to decline earlier in mev-1. Thus the average life span of mev-1 mutants is only 9.4 days compared to 14.3 days for the wild type.
,
O
J
'1-
2
4
6
DAYS A F T E R
8
10
OXYGEN
12 0
HATCH
lO0 -
_
Z0
~
C O N C E N T R A T I O N (%)
40
6• ;
80 100 - , "--,
Fig. 2. The number of eggs laid by mev-I and wild-type hermaphrodites. Ten animals were observed for the wild type and 15 for rnev-1. Vertical lines represent standard deviations.
mutants survived (Fig. 1). Comparing the concentrations causing 40% lethality, the mev-1 mutants are about 4 times more sensitive than the wild type.
z
9 o iJ.
Fecundity The mev-1 mutants have normal morphology. However, they have low vital activity, i.e., slow growth and low fecundity. Egg-laying in the wild type begins on the third day after hatching, reaches a m a x i m u m of about 130 eggs per animal per day on the fifth day and continues for another week (Fig. 2). The average number of eggs laid per individual was 287 + 34 (n = 10). The onset of egg production was delayed for about half a day in mev-1 mutants and the average number of eggs laid per animal was only 77 + 48 (n = 15). Life span A typical survival curve is shown in Fig. 3. The m a x i m u m life span (21 days) is nearly the same for rnev-1 mutants and the wild type but survival
10 z
!
Fig. 4. Sensitivity of mev-1 mutants to oxygen gas. L~ larvae were exposed to oxygen/nitrogen mixtures at various concentrations and their survival was scored after 3 days. Vertical bars represent standard deviations from 4 replicate experiments. Approximately 100 animals were scored for each strain. Closed circles, wild type; open circles, mev-1.
169 TABLE 1 SOD ACTIVITY IN T H E mev-I A N D WILD TYPE OF C.
elegans Strain
Activity a (units)
Mn-SOD b (%)
Wild type
77.8 90.0 39.2 41.6
almost 0 almost 0 14 21
mev-1
a Data from 2 experiments are shown as units per mg protein. b Percent activity insensitive to 1 mM KCN of total activity.
Sensitivity to oxygen gas Interestingly, mev-1 mutants are even more hypersensitive to oxygen gas than to methyl viologen (Fig. 4). Whereas wild-type animals can develop nearly normally under 90% oxygen, few mev-1 larvae survive a 3-day exposure. The growth and movement of mev-1 larvae were nearly normal for the first day of exposure, but arrested thereafter. SOD activity Superoxide dismutase in eukaryotic cells is composed of 2 types, one containing Zn and Cu in the active center and another containing Mn (Halliwell and Cutteridge, 1985). The activities of these 2 types can be distinguished by adding 1 m M KCN to the reaction mixtures, which specifically inhibits the Z n / C u enzyme. The SOD activity in mev-1 is about 50% of that in the wild type (Table 1). Most wild-type activity is inhibited by KCN, indicating that there is relatively little, if any, Mn-SOD. In contrast, 14-21% of the residual activity in mev-1 could be attributed to the Mn-SOD. As a caveat, these measurements were made from asynchronous populations and are conceivably affected by unrecognized differences in the stage distributions of the populations. Discussion
A C. elegans mutant was isolated which is about 4 times more sensitive to methyl viologen (paraquat) than the wild type. The gene was designated mev-1. Genetic analysis revealed that the mev-1 mutation is located on linkage group III.
These mutants have low fecundity and consequently populations grow slowly. As there are other C. elegans mutants with small brood sizes, this is not a specific property of mev-1 (Swanson et al., 1984). The mev-1 mutants were found to be hypersensitive to oxygen gas as well as methyl viologen and have reduced Z n / C u - S O D activity. This suggests that the toxicity of methyl viologen is mediated, at least in part, by its ability to generate superoxide anions (Bagley et al., 1986; Krall et al., 1988) and that the resistance of wildtype nematodes to methyl viologen is mediated by SOD (Hassan and Fridovich, 1978). Methyl viologen-sensitive or oxygen-sensitive mutants have been isolated in various organisms, including E. coli (Hassan and Fridovich, 1979; Fridovich, 1986; Carlioz and Touati, 1986), B. coagulans (Vassilyadi and Archibald, 1985), yeast (Bilinski et al., 1985; Van Loon et al., 1986) and Drosophila melanogaster (Tang et al., 1986; Phillips et al., 1989). In several cases, these mutants were defective in SOD (Hassan and Fridovich, 1979; Fridovich, 1986; Carlioz and Touati, 1986; Bilinski et al., 1985; Van Loon et al., 1986), but oxygen hypersensitivity need not be accompanied by reduced SOD activity (Carlioz and Touati, 1986). With possible rare exceptions, all known oxygen-tolerant organisms have SOD (Fridovich, 1983), suggesting that SOD could provide some oxygen tolerance to most organisms. SOD-defective mutants have been isolated recently in D. melanogaster (Tang et al., 1986; Phillips et al., 1989). One type of mutant (Tang et al., 1986) was found to be hypersensitive to X-rays and another type (Phillips et al., 1989) was completely defective in Cu/Zn-SOD, had a very short life span and was sensitive to methyl viologen. But sensitivity to oxygen was not examined for either type of mutant. Our mev-1 data are possibly the first mutant studies to suggest a correlation between oxygen or methyl viologen sensitivity and SOD activity in animals. In humans, 21-monosomy reduces SOD activity by about half compared to normal controls (Yoshimitsu et al., 1983; Nakai et al., 1984). In contrast, 21-trisomy (Down's syndrome) may result in overexpression of the SOD gene (Groner et al., 1986; but see Miyazaki et al., 1987; Jeziorowska et al., 1987). It has been pro-
170
posed that SOD overexpression may have an etiological role in Down's syndrome. Like the cSOD "1°8 mutant of Drosophila (Phillips et al., 1989), a characteristic of mev-1 strains is a shortened life span. The average life span of mev-1 animals is only 66% of the wild-type life span. The life span of the wild type remained constant over the range of 2-40% oxygen, while the life span of the mutant varied in dependence on the concentrations of oxygen (unpublished). We suggest that the reduced life span is caused by oxygen damage, and in particular by superoxide anions. The reduced life span could reflect an acceleration of normal aging processes. In any case, SOD appears to be required for a normal life span in C. elegans. According to Cutler (1984), the concentration of various endogenous antioxidants such as SOD, urate, or vitamin E, is positively correlated with the longevity of mammals, including primates. For SOD, however, the activity divided by the standard metabolic rate, rather than the SOD activity itself, is more linearly related to the maximum life span (Tolmasoff et al., 1984). Since we have not examined the standard metabolic rate of mev-1 or wild-type nematodes, comparison at this level is not yet possible. There is a recent report by Morimyo (1988) which deals with a methyl viologen-sensitive mutant of E. coli (mvr). However, the cloned gene did not hybridize with any of the cloned SOD genes (personal communication). Regardless of the structure and function of the mev-1 gene, this is the first report which deals with defective SOD activity in C. elegans.
Acknowledgements We are indebted to Dr. E.M. Hedgecock, Department of Biology, Johns Hopkins University and Dr. T. Johnson, University of Colorado, for their kind advice and encouragement during this study and for reading the manuscript. References Bagley, A.C., J. Krall and R.E. Lynch (1986) Superoxide mediates the toxicity of paraquat for Chinese hamster ovary cells, Proc. Natl. Acad. Sci. (U.S.A.), 83, 3189-3193.
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Tolmasoff, J.M., T. Ono and R,G. Cutler (1984) Superoxide dismutase: correlation with life-span and specific metabolic rate in primate species, Proc. Natl. Acad. Sci. (U.S.A.), 77, 2777-2781. Van Loon, A.P.G.M., B. Pesold-Hurt and G. Schatz (1986) A yeast mutant lacking mitochondrial manganese-superoxide dismutase is hypersensitive to oxygen, Proc. Natl. Acad. Sci. (U.S.A.), 83, 3820-3824. Vassilyadi, M., and F. Archibald (1985) Catalase, superoxide dismutase, and the production of O2-sensitive mutants of Bacillus coagulans, Can. J. Microbiol., 31, 994-999. Yoshimitsu, K., S. Hatano, Y. Kobayashi, Y. Takeoka, M. Hayashidani, K. Ueda, K. Nomura, K. Ohama and T. Usui (1983) A case of 21q-syndrome with half normal SOD-1 activity, Hum. Genet., 64, 200-202.
