Mutagenicity of a bipyridylium herbicide in an N2-fixing cyanobacterium Nostoc muscorum

Environment International, Vol. 10, pp. 285-290, 1984 0160-4120/84 $3.00 + .00 Printed in the USA. All rights reserved. Copyright © 1985 Pergamon Press Ltd.

MUTAGENICITY OF A BIPYRIDYLIUM HERBICIDE IN AN N2-FIXING CYANOBACTERIUM NOSTOC MUSCORUM

A. Vaishampayana Department of Botany, R.L.S.Y. College, (Magadh University), Bakhtiyarpur-803212 (Patna), India

(Received 28 March 1984; Accepted 4 September 1984)

The herbicide diquat (1,1'-ethylene-2,2'-bipyridylium ion) is toxic and lytic to Nostoc muscorum. A reverse mutation (from her nil- auxotrophy to het ÷ n(f ÷ prototrophy), a forward mutation (for streptomycin [St]-resistance), and an auxotrophic mutation (carbon-auxotrophy through methylamine [MA]-resistance) have been obtained with a dose of diquat permitting about 20070 or 50070 survival. Similar mutation frequencies were obtained through induction with MNGG (N-methyl-N'-nitro-N- nitrosoguanidine).

Introduction

During the last decade the mutagenicity of a large number of pesticides and food additives have been detected in bacterial, mammalian, and higher plant systems (Drake et al., 1975; Shirasu et al., 1976; Ames, 1979; DeSerres, 1979; Drake, 1980; Griffiths, 1981; Haroun and Ames, 1981; Nagao et aL, 1981; Sugimura et aL, 1981; VanVlett and Reinhardt, 1983). Of these, the pesticides belonging to bipyridyl group of synthetic organic chemicals are able to liberate free radicals during interactions with biological systems (Moody and Hassan, 1982); they have been reported to induce mutations in the spring onion All iumf is tu losum (Alekperew, 1967), in hens for the production and viability of eggs (Fletcher, 1967) and in yeast for the mitotic gene conver- sions (Parry, 1973).

These studies determined that bipyridyl compounds might induce genetic changes in microbes of agricultural importance, including soil bacteria and cyanobacteria (Parry, 1973; Vaishampayan, 1979, 1984a,b). The nitrogen-fixing cyanobacteria of the paddy fields (Singh, 1961; Fogg, 1974; Venkataraman, 1981)which remain in natural contact with many of the agrochem- icals in practice (Vaishampayan et aL, 1978; Vaisham- payan and Prasad, 1981a, 1982, 1984; Vaishampayan, 1984c,d,e) have recently been found to be good in-

aAuthor's mailing address: Private Mail Bag, Chaturbhujasthan, Muzaffarpur-842001, India

dicator organisms for pesticide-screening purposes in relation to environmental mutagenesis (Singh and Vaishampayan, 1978; Singh et al., 1979; Vaishampayan and Prasad, 1981b; Vaishampayan, 1984a, b). Yet, such studies are limited, especially as far as the mutagenicity screening of bipyridyl chemicals in this system is con- cerned.

Diquat is a bipyridyl herbicide, popularly used in the paddy fields of North India; its effect has not yet been noted for any cyanobacterium. This prompted the author to investigate whether this chemical can induce all the three possible types of mutation, as suggested by Zimmermann (1973), i.e., reverse, forward, and auxo- tropic mutations, for placing it in the category of potent mutagens for cyanobacteria. As such, during the present study the mutagenicity of diquat has been worked out in different strains of a cyanobacterium Nostoc muscorum, using the following markers on which spontaneous mutational work has been previously conducted: (1) reverse mutation from het- nif- auxotrophy to het ÷ n i f ÷ prototrophy (Singh and VaJshampayan, 1978); (2) forward mutation for resistance to 10 #g/mL St (0.30 #g/mL St is toxic to N. muscorum) (Stewart and Singh, 1975); (3) auxotrophic mutation for carbon-requirement through 5 mM MA-resistance (1 mM MA is toxic to N. muscorum) (Vaishampayan, 1982a, 1983a). These effects have been compared with those of the well-known mutagen MNNG, which belongs to nitrosamine group but was selected as a positive control because (1) it is a potent mutagen in cyanobacteria (see Herdman, 1982),

285

286 A. Vaishampayan

and (2) diquat possesses reactive ethylene group and may be capable of alkylating DNA (Drake and Baltz, 1976), similar to the activity of MNNG.

Materials and Methods

Organisms The filamentous, heterocystous, N2-fixing (het ÷ n i f +)

parent Nos toc m u s c o r u m and its nonheterocystous, non-N~-fixing mutant (het- nif-) strains (obtained from H. N. Singh's personal culture collections, Central University of Hyderabad, India) were used as test materials. They were chosen because of their suitability in physiological, genetic, and mutational studies (Singh et al., 1978a,b; Vaishampayan and Singh, 1981a,b; Vaishampayan, 1979, 1983b, 1984f, g).

Culture condi t ions and assessments The her ÷ n i f ÷ parent and the het- ni f - mutant strains

of N. m u s c o r u m were grown routinely in modified Chu 10 medium (Gerloff et al., 1950) at a temperature of 28 a:2 °C and a continuous fluorescent light flux of 10 W m -2 (400 to 700 nm), after Vaishampayan (1982b). Growth of the experimental cultures (homogeneous sus- pensions) was measured colorimetrically at 663 nm (A 663) during the optimum growth phase, i.e., on tenth day of inoculation (on completion of 8 doubling times; the generation or doubling time of the two cyanobacterial strains is 27 h) (Vaishampayan, 1981).

Heterocyst frequency was assessed microscopically in terms of number of heterocysts per hundred vegetative cells, according to the method described by Vaisham- payan (1982c). Under conditions of massive fragmenta- tion heterocyst frequency was assessed as number of heterocysts per hundred vegetative cells in a microscopic field (Vaishampayan, 1983c).

Physiological e f fects o f the herbicide A log-phase culture of both het ÷ n i f ÷ and het- ni f -

strains of N. m u s c o r u m was harvested, washed re- peatedly with sterile glass-distilled water and presus- pended in N2 (combined nitrogen-free) medium to an OD of 0.25. Two mL aliquots were then inoculated into 50 mL of fresh sterile N~ and NO~ (5 mM) media, unsupplemented or supplemented with different concentrations (0, 60, or 90, /zg/mL) of diquat. The experimental cultures were incubated in the growth chamber and their optimum growth as well as maximum heterocyst frequency were recorded on tenth and third day of inoculation, respectively.

Mutagenici ty screening o f the herbicide For mutagenicity studies, the effects of diquat as well

as the known mutagen MNNG on the survival of the parent organism were examined, following the method of Singh and Vaishampayan (1978). The exponentially growing liquid-mediated organism was harvested, frag-

mented into unicells (Vaishampayan and Prasad, 1982) and treated for 20 min with various concentrations of the two chemicals (made by appropriate dilutions with sterile glass-distilled water). The treated organism and the untreated control were washed and spread over different sets of the solid (1.2%) difco bacto agar N2 medium. After 14 days of incubation in the growth chamber, viable colonies appeared from amongst the variously treated cyanobacterial samples, the number of which was counted and compared with the control to determine percentage survival (Singh and Vaisham- payan, 1978). Afterwards, 20% and 50% survival doses of the two chemicals were used (in the manner used during treatments for scoring the survival) to induce mutations for (1) reversion from het- ni f - auxotrophy to bet ÷ n i f ÷ prototrophy, (2) resistance to 10 #g/mL St, and (3) carbon-auxotrophy through 5 mM MA-resistance.

The parent and the het- ni f - mutant strains of N. m u s c o r u m were fragmented into unicellular bits. General methods for mutagenesis assays (selective plate techniques in N. muscorum) were used (Singh and Vaishampayan, 1978; Stewart and Singh, 1975; Vai- shampayan, 1982a), with the exception that diquat-and MNNG- treated samples were run concurrently to make comparative assessments. The stability of a certain number of putative mutant colonies formed after 14 days of incubation was tested for phenotypic characteristics as follows.

A putative het ÷ n i f ÷ revertant colony of the bet- ni f - mutant was raised to clonal culture in N2 medium. Ali- quots from this stock were inoculated into both solid and liquid N2 and NO; media. Ten subsequent transfers were made in the two nitrogen media in both solid as well as liquid growth conditions. Since chances of instability were greater in the NO; medium, the progeny of No~-mediated cultures was tested after each transfer for their heterocyst frequency and growth efficiency in N2 medium. Stability was maintained for those population that consistently exhibited a significant heterocyst- forming capacity and growth in N2 medium after each transfer.

Similarly, many other colonies were examined. A putative St r mutant colony was grown into a clonal culture in the N2 medium. Since chances of instability were greater in a streptomycin-lacking medium, the progeny from the streptomycin-lacking cultures were tested after each transfer for their growth with 10/~g/mL streptomycin. Stability was maintained for the population that consistently exhibited a significantly normal growth with 10 #g/mL streptomycin after each transfer.

Finally, a putative M A r mutant colony was raised to clonal culture in 5 mM MA-containing N2 medium. Ali- quots from this stock were inoculated into both solid and liquid N2 media containing or lacking 3 mM glucose or 5 mM MA. Ten subsequent transfers were made in the same manner. The progeny line (tested after each transfer) that grew even in the absence of glucose or MA

Mutagenicity of a bipyridylium herbicide 287

Table 1. Data a on N2- and NO;-mediated growth b and heterocyst frequency c of Nostoc muscorum strains: (i) het ÷ n ip parental strain, (ii) het- nif- mutant, (iii) M A r mutant, (iv) St r mutant, and (v) het* ni f ÷ revertant of the het- nif- mutant, unsupplemented or supplemented with diquat or other substrate.

Growth Heterocyst

Strain Supplement Concentration N2 NO; Frequency (N2)

(i) nil nil 0.635 ±0.010 0.705 ±0.012 5.6 ±0.8 (i) diquat 60 #g/mL 0.106 ±0.013 0.130 ±0.014 5.4 ±0.4 (i) do 90/~g/mL 0.0 0.0 5.4 ±0.3 (i) MA 0.5 mM 0.301 ±0.025 0.324 ±0.019 1.2 ± 1.0 (i) do 1.0 mM 0.0 0.0 0.0 (i) St 0.25 #g/mL 0.273 ±0.011 0.285 ±0.017 5.3 ±0.8 (i) do 0.30/zg/mL 0.0 0.0 5.3 ±0.2

(ii) nil nil 0.0 0.630 ±0.017 0.0 (ii) diquat 60/zg/mL 0.0 0.115 ± 0.016 0.0 (ii) do 90 #g/mL 0.0 0.0 0.0

(iii) nil nil 0.0 0.0 3.6 ±0.5 (iii) MA 5 mM 0.645 ±0.020 0.650 ±0.115 0.0 (iii) glucose 3 mM 0.590 ±0.015 0.635 ±0.010 5.2 ±0.4

(iv) nil nil 0.525 ±0.011 0.570 ±0.018 5.4 ±0.3 (iv) St 10 #g/mL 0.520 ±0.016 0.585 ±0.014 5.5 ±0.6

(v) nil nil 0.480 ±0.012 0.505 ±0.012 4.7 ±0.8 (v) diquat 60 #g/mL 0.090 ±0.012 0.107 ±0.011 4.7 ±0.5 (v) 90 #g /mL 0.0 0.0 4,7 ±0.2

aThe values are the means of five independent readings with their respective standard errors. blncrease in OD on tenth day of inoculation (A66~); initial OD in each case was 0.005. cThe maximum value was obtained on third day of inoculation; no heterocyst was formed in NO] medium.

was discarded; stability was maintained for the one that consistently exhibited an exclusive requirement for an exogenous supplementation of either glucose or MA for growth on both solid and liquid media. Other colonies were also examined.

Chemica l C o m p o u n d s

MNNG, streptomycin, glucose, and methylamine (obtained from Sigma Chemical Co., St. Louis, MO) were filter sterilized, following the method of Vaisham- payan and Hemantaranjan (1984), and then added to the already sterilized (autoclaved) and cooled mineral medium. All other chemicals (medium constituents) were obtained from British Drug House, Poole, Dorset, England. The herbicide was a gift from the Lakshami Chemicals Pvt. Ltd., Phatuha, Patna, India.

Results and Discussion

At a concentration of 90/zg/mL and above, diquat proved to be growth-inhibitory and lytic (within 48 h association) to the het ÷ n i f ÷ and her- n i f - strains of N o s t o c m u s c o r u m in liquid (Table 1) as well as solid (Table 2) N2 and/or NO; medium, without any adverse effect on the formation of heterocysts specifically in N2 medium (Table 1). (Note that in Table 2, data are not provided for the her- ni f - strain due to approachingly

similar pattern of survival to the het ÷ n i f ÷ strain.) Ob- viously, these effects of diquat are due neither to the in- hibition of photosynthesis or protein synthesis, nor to the nitrogen metabolism. It is known that the presence

Table 2. Data a on percent survival of parent Nostoc muscorum treatedb with various concentrations of diquat and MNNG in N2 medium.

Concentration of the Chemicals

(#g/mL)

Percent Survival (o70)

Diquat Treated MNNGTrea ted

10 73.15 ±0.22 89.36 +0.28 20 62.63 + 1.77 83.82 + 1.64 30 55.48 ± 1.4.4 83.78 + 1.45 40 49.15 ±2.19 82.36 ±1.25 50 35.33 +3.08 82.03 +6.17 60 28.25 ±1.39 79.55 ±2.32 70 19.92 ±1.07 74.42 ±0.34 80 7.63 + 1.26 67.85 ± 1.45 90 0.0 67.32 ±2.41

100 0.0 61.40 ± 1.36 110 0.0 57.38 ±2.44 120 0.0 50.02 ± 1.13 130 0.0 42.35 ±3.22 140 0.0 34.02 ±3.66 150 0.0 21.37 ±2.69

aThe values are the means of five independent readings with their respective standard errors. bTreatment time was 20 min in each case.

288 A. Vaishampayan

of heterocysts in N2 medium is the indicator of active photosynthesis and protein synthesis (Singh and Vai- shampayan, 1978); their absence in NO; medium in- dicates active NO; metabolism (Singh e t al. , 1977). However, the lytic effects of diquat were found similar to those in the higher plants where the bipyridylium ion is reported to release a free radical on receiving an elec- tron from photosystem I (Hall, 1976) which is reoxidized by molecular oxygen to form hydrogen peroxide. This hydrogen peroxide liberates hydroxy, and also possibly peroxy, free radicals (Dodge, 1975) which show their abrupt phytotoxic action by rapid membrane disruption (Brian e t al. , 1958; Dodge, 1971, '75; Mees, 1960) leading to mutation induction (Moody and Hassan, 1982).

In consonance with the above situation, the three categories of mutation (reverse, forward, and auxotrophic) could be significantly scored (Table 3) on induction with the 20070 and 50070 survival doses of diquat as well as MNNG (70 and 40 #g/mL for diquat; 150 and 120 #g/mL for MNNG, respectively, treated for 20 min each; see Table 2). The genetic changes were found true to characterization for the phenotypic expressions. For example, (1) the b e t ÷ n i f ÷ prototrophic revertants of the b e t - n i f - strain of N o s t o c m u s c o r u m were found to have "parent-like" N2-mediated growth and heterocyst- forming characteristics (Table 1). Thus they were quite similar to such spontaneous revertants studied previously (Singh and Vaishampayan, 1978). (2) The S t r mutants

of N . m u s c o r u m were found to be resistant to 10 #g/mL St (Table 1). Thus they were quite similar in this respect to the spontaneous S t r mutants of this organism isolated previously (Stewart and Singh, 1975). (3) The M A r

mutants of N . m u s c o r u m were found to use 5 mM MA or 3 mM glucose (Table 1) as an essential growth factor. Thus they were quite similar in this respect to the spontaneous M A r (carbon-requiring) mutants of N . m u s -

c o r u m isolated previously (Vaishampayan, 1982a, 1983a).

Since diquat caused all the possible types of mutagenicity as suggested by Zimmerman (1973) for placing it in the category of a potent mutagen, it may be concluded that this chemical is an effective mutagen for cyanobacteria. Furthermore, since diquat-induced mutation frequencies for all the three markers were well comparable to those obtained through MNNG-induction (Table 3), diquat definitely appears to be a powerful mutagen in this group of photosynthetic microorganisms. Although free radicals have been shown to have mutagenic properties, mutational yields of bipyridylium ions are much lower in microorganisms and yeast (Parry, 1973; Vaishampayan, 1979; Yamanaka, 1983) compared to those in plant organisms where reduction and radical formation can take place to a greater extent (Dodge, 1971, 1975).

The observed mutational yield of diquat was higher in the cyanobacterium N o s t o c m u s c o r u m than in other microbes (Vaishampayan, 1979). This may be correlated

Table 3. Data a on spontaneous and induced (by 2007o and 50070 survival doses of diquat and MNNG) b mutation frequencies for (i) reversion of het- ni f - mutant to bet ÷ n(f* prototrophy, (ii) 10 ~g/mL St r, and

(iii) 5 mM M A r leading to carbon-auxotrophy in Nostoc muscorum.

Frequency of Colonial Growth on Mutant Number of Colonies

Induced By: Marker Selection Medium Tested for Stability c

nil (spontaneous) (i) 2.4 4. 1.5 x 10-' 12 (8) nil (ii) 1.2 4. 0.3 x 10 -9 16 (12) nil (iii) 2.5 4. 2.4 x 10 -~ 14 (11)

70/zg/mL diquat (i) 2.2 4. 0.5 x 10 -3 50 (38) 70/zg/mL diquat (ii) 2.7 4. 1.1 x 10-" 37 (25) 70 #g/mL diquat (iii) 3.1 4. 0.5 x l0-" 39 (30)

40 ~g/mL diquat (i) 0.2 4. 1.1 x 10 -3 32 (21) 40 / tg /mL diquat (ii) 1.8 4. 0.9 x 10-" 41 (29) 40/~g/mL diquat (iii) 2.5 4- 1.6 × 10 -4 45 (33)

150 ~g/mL MNNG (i) 4.7 ± 1.2 x 10 "3 22 (17) 150 ~g/mL MNNG (ii) 5.8 ± 2.2 x 10 -s 33 (27) 150/~g/mL MNNG (iii) 5.1 ± 1.6 x 10 -4 29 (22)

120/~g/mL MNNG (i) 3.6 ± 1.8 × 10 -3 29 (22) 120 ~g/mL MNNG (ii) 3.8 ± 1.7 × 10 -s 32 (25) 120/~g/mL MNNG (iii) 3.5 ± 2.3 x 10 -4 30 (24)

aThe values are the means o f five independent readings with their respective standard errors. bDifferent concentrations of the two chemicals have been used in order to examine whether the compounds are capable enough to induce various mutations at the two different survival doses. CNumber o f colonies found stable, has been given in parentheses after each value.

Mutagenicity of a bipyridylium herbicide 289

with the fact that, unlike in other microorganisms, photosynthesis in cyanobacteria resembles that found in the chloroplasts of higher plants, involving reduction and radical formation at a similar rate (Stewart et al., 1975). Reasonably, therefore, this accounts for the observed diquat-induced rapid membrane disruption and cellular lysis as well as the creditably higher mutational yields in the surviving cells of N. m u s c o r u m involving a wider range of all possible mutations. On the other hand, the diquat-induced mutation frequenceis obtained in this study are comparable to an MNNG- induced mutation frequency in Escherichia coil K12, where auxotrophic mutations in over 40070 and valine- resistant mutation in about 0.2070 of the surviving cells have been reported, although at different loci (Adelberg et al., 1965). Since diquat, like MNNG, possesses an alkylating group it is doubted whether it might act like MNNG to some extent. In any event, the higher mutational yield obtained through diquat treatment in N. muscorum, most probably due to the action of bipyridylium ions, or a combination of both bipyridyl and alkyl groups, may be strongly favored.

Further detailed biochemical studies on the mode(s) of action of diquat, as well as in vitro herbicide-microbe interactions, are in progress in an attempt to establish the strong mutagenicity of this bipyridyl herbicide. This agricultural chemical has conferred great benefits in in- creasing paddy yields per hectare and per man hour in North India (Ram, 1976). However, in view of the present results, its increased discharge into the environment leads to serious concern regarding the possible toxic hazards to a living system. Thus, there is wide scope for thorough investigation of the tropical rice-fields for in- digenous flora of these self-renewable N2-fixing cyanobacteria which have been consistently receiving diquat treatment for the last few years. Nevertheless, such chemicals may also prove deleterious to the main crop (paddy) where oxygenic photosynthesis is even more rapid (Murata, 1964). There may be possible carcinogenic hazards to mankind, since many mutagens are reportedly carcinogenic to mammalian systems, causing tumors and cancers (Ames, 1979).

Acknowledgements-The author wishes to thank Girish Nath, Pro- prietor, Lakshami Chemicals Pvt. Ltd., Phatuha (Patna), India, for providing the sample of the herbicide for the present investigation. Thanks are also due to the author's wife, Anupama Sahay, for care- fully reading and commenting upon the manuscript as well as for her skilled and excellent technical assistance. Financial assistance for this work in the form of a highest grade of Research Associateship Award to the author from the University Grants Commission, Government of India, New Delhi-ll0002 (Grant No. F/16-51/83) is gratefully acknowledged.

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Mutagenicity of a bipyridylium herbicide in an N2-fixing cyanobacterium Nostoc muscorum