THE RADIO SENSITIZING EFFECT OF METRONIDAZOLE IN MAIZE

NOTA

THE RADIOSENSITIZING EFFECT OF METRONIDAZOLE IN MAIZE (¹)

LYDERSON FACIO VICCINI(²), LUIZ SÉRGIO SARAIVA(³), JOSÉ DE ALMEIDA FILHO(³), COSME DAMIÃO CRUZ(³) and ROGÉRIO ALVAREZ DE ANDRADE(³)

ABSTRACT

The identification of chemical substances which increase the efficiency of radiation is important to make easier the obtention of plants with structural chromosome aberrations which may be used in an alternative program for hybrid maize production. The present work was carried out to investigate the effect of the chemical substance metronidazole in maize seedlings submitted to gamma radiation. Several treatments were done, soaking the seeds in solutions with varied concentrations of the active substance combined with solution filtration and gamma radiation. On the third day of the experiment, germination percentage, root and stem lengths were evaluated. At a high concentration (1,250 mg/50 mL) metronidazole behaved as a radiosensibilizer in the presence of radiation. Even at a low concentration (250 mg/50 mL; 750 mg/50 mL) and in the absence of radiation, metronidazole behaved as toxic substance.

Index terms: corn, Zea mays, gamma radiation, metronidazole.

RESUMO

EFEITO RADIOSSENSIBILIZADOR DO METRONIDAZOL EM MILHO

A identificação de substâncias químicas que aumentam a eficiência da radiação é importante para facilitar a obtenção de plantas portadoras de aberrações cromossômicas estruturais que possam ser usadas em um programa alternativo de produção de milho híbrido (Zea mays L.). Realizou-se o presente trabalho com o objetivo de investigar o efeito da substância química metronidazol na resposta de plântulas de milho à radiação gama. Diversos tratamentos foram feitos, embebendo as sementes em soluções com variadas concentrações da substância ativa, combinadas com filtragem e radiação gama. No terceiro dia do experimento, foram avaliados a porcentagem de germinação e os comprimentos das raízes e dos caules. Em alta concentração (1.250 mg/50 mL), o metronidazol, em presença de radiação, comportou-se como radiossensibilizador e, na sua ausência, como substância tóxica, mesmo em baixa concentração (250 mg/50 mL e 750 mg/50 mL).

Termos de indexação: milho, Zea mays, radiação gama, metronidazol.

Radiation has been extensively used to induce mutations (Smith, 1958; Ananthaswamy et al., 1969; Iqbal & Zahur, 1975; Gorla et al., 1987; Muhanna et al., 1991). It can also produce structural chromosome aberrations (Kowyama et al., 1984; Pimentel, 1990; Cuny et al., 1993). Mutations, however, do not cause only harmful effects and plant breeders showed that they were also an additional tool in the search for more desirable cultivars (Bouvier et al., 1993; Maluszynski et al., 1995). Several studies proposed the use of structural chromosome aberrations in hybrid maize production combined with genetic male-sterility (Ramage, 1965; Patterson(⁴); Doyle, 1972).

The use of higher doses of radiation to obtain the desired aberrations with greater ease, may however, be lethal to the plants. The identification of chemical substances which increase the radiation efficiency is an option to facilitate the obtention of the desired aberrations. Several substances have been successfully investigated, including "timine" analogs (Hall, 1982), N-methyl-nitrosourea (Olejniczak, 1986, 1987), sodium "azide" (Cheng & Gao, 1988) and mebendazole (Santos, 1993). The present study was carried out to investigate the effect of the chemical substance metronidazole on the response of maize to gamma radiation.

MATERIAL AND METHODS

Maize seeds of the breeding line L-SRR-D-86-19 from the Federal University of Viçosa were soaked for 24 hours in a solution of metronidazole at concentrations of 250 and 750 mg/50 mL. In a second experiment, the seeds were soaked in a more concentrated solution (1,250 mg/50 mL) for 24 hours.

Preliminary tests indicated the best dose to be used. After soaking, the seeds were exposed to 70 Gy of gamma radiation, emitted by a Co⁶⁰ source. They were then distributed in Petri dishes and placed in an incubator at a temperature of 30°C. Each treatment was made up of 52 seeds divided in four replications (each Petri dish corresponding to one replication).

The tablets containing the active substance were squashed in distilled water to dissolve and the volume was then completed to 50 mL to prepare the solutions.

The seeds were soaked in filtered and non-filtered solutions, in order to investigate the influence of the "incipient" contained in the tablets.

The treatments for the experiment involving soaking in a metronidazole solution at the concentrations of 250 and 750 mg/50 mL were: 01. Seeds soaked in metronidazole solution (250 mg/50 mL), non-filtered, non-irradiated; 02. Seeds soaked in metronidazole solution (750 mg/50 mL), non-filtered, non-irradiated; 03. Seeds soaked in metronidazole solution (250 mg/50 mL), non-filtered, irradiated with 70 Gy; 04. Seeds soaked in metronidazole solution (750 mg/50 mL) non-filtered, irradiated with 70 Gy; 05. Seeds soaked in metronidazole solution (250 mg/50 mL), filtered, non-irradiated; 06. Seeds soaked in metronidazole solution (750 mg/50 mL), filtered, non-irradiated; 07. Seeds soaked in metronidazole solution (250 mg/50 mL), filtered, irradiated with 70 Gy; 08. Seeds soaked in metronidazole solution (750 mg/50 mL), filtered, irradiated with 70 Gy; 09. Seeds soaked in distilled water, non-irradiated and 10. Seeds soaked in distilled water, irradiated with 70 Gy.

For the experiment with soaking in solution at the concentration of 1,250 mg/50 mL, the treatments were: 01. Seeds soaked in a metronidazole solution (1,250 mg/50 mL), non-filtered, non-irradiated; 02. Seeds soaked in metronidazole solution (1,250 mg/50 mL), non-filtered, irradiated with 70 Gy; 03. Seeds soaked in metronidazole solution (1,250 mg/50 mL), filtered, non-irradiated; 04. Seeds soaked in metronidazole solution (1,250 mg/50 mL), filtered, irradiated with 70 Gy; 05. Seeds soaked in distilled water, non-irradiated and 06. Seeds soaked in distilled water, irradiated with 70 Gy.

Germination percentage (GER), root length in centimeters (RAD), and stem length (CAU) in centimeters, were assessed on the third day of the experiment.

A preliminary test for cytological effect of the metronidazole was done through analysis of pollen fertility, that is a sensitive test to detect chromosome aberration. That being so, under field conditions, the M2 generation obtained through self-pollination from the M1 plants was analyzed. The M1 plants were obtained from seeds soaked in a metronidazole solution (250 mg/50 mL) irradiated with 70 Gy of gamma radiation and also control plants without irradiation. Fifty seeds were planted in two rows, with twenty five seeds per row.

The statistical analysis of the laboratory experiments was carried out splitting the sum of the squares for treatments in orthogonal contrasts. The contrasts between treatment means (m) were based on the selected comparisons and tested by the F test at the 5% and 1% levels of probability. For 250 mg/50 mL and 750 mg/50 mL concentrations the contrasts were used as follows: contrast 1 was used to test radiation effect (m3 + m4 + m7 + m8 + m10) vs. (m1 + m2 + m5 + m6 + m9); contrast 2 was used to test metronidazole effect in presence of radiation (m3 + m4 + m7 + m8) vs. (4m10); contrast 3 was used to teste the effect of the metronidazole concentration with radiaton (m3 + m7) vs. (m4 + m8); contrast 4 was used to test filtering effect in 250 mg/50 mL concentration with radiation (m7) vs. (m3); contrast 5 was used to test filtering effect in 750 mg/50 mL concentration with radiation (m4) vs. (m8); contrast 6 was used to test toxic effect of metronidazole without radiation (m1 + m2 + m5 + m6) vs. (4m9); contrast 7 was used to test the effect of the concentration of metronidazole without radiation (m1 + m5) vs. (m2 + m6); contrast 8 was used to test filtering effect in 250 mg/50 mL concentration without radiation (m1) vs. (m5); contrast 9 was used to test filtering effect in 750 mg/50 mL concentration without radiation (m2) vs. (m6).

For 1,250 mg/50 mL concentration, the contrasts were used as follows: contrast 1 was used to test radiation effect (m2 + m4 + m6) vs. (m1 + m3 + m5); contrast 2 was used to test the radiosensitizing effect of metronidazole (m2 + m4) vs. (2m6); contrast 3 was used to test filtering effect with radiation (m2) vs. (m4); contrast 4 was used to test toxic effect of metronidazole without radiation (m1 + m3) vs. (2m5); contrast 5 was used to test filtering effect without radiation (m1) vs. (m3).

Descriptive statistical analysis was used for the field data.

RESULTS AND DISCUSSION

Table 1 shows the statistical analysis of the traits GER, RAD and CAU, for the concentrations at 250 mg/50 mL and 750 mg/50 mL. Significant differences were found among the treatments for all the traits. The splitting of the treatments sum of the squares was carried out using orthogonal contrasts, so that the significance could be studied in detail. To clarify treatments behavior, Table 2 shows the means of the three traits analysed.

The harmful effect of radiation (C1) was confirmed from the contrasts studied for the characteristics RAD and CAU. The effect of the metronidazole solution was studied both in the presence and absence of radiation, by the contrasts 2 and 6, respectively. For the concentrations at 250 and 750 mg/50 mL, no radiosensibilizing effect of the metronidazole was found (C2). However, a toxic effect was detected in the absence of radiation (C6).

The effect of filtering on the solution was studied by the contrasts 4 (soaking in solution at 250 mg/50 mL and irradiation), 5 (soaking in solution at 750 mg/50 mL and irradiation), 8 (soaking in solution at 250 mg/50 mL without irradiation) and 9 (soaking in solution at 750 mg/50 mL without irradiation). Significances were not observed for any traits in contrasts 4 and for few traits in contrasts 5 and 8, while in contrast 9, there was significance for all the traits analyzed. This may be due to the presence of Petri dishes contaminated by fungi, which developed because of the following suitable conditions: a) a quantity of starch placed at the bottom of the dish, b) the favorable temperature and moisture, and c) presence of harmed the seeds. This problem probably appeared in contrast 9 due to the lack of radiation and the high concentration of compost used. In contrast 5, where the concentration was the same as in contrast 9, the radiation may have caused enough damage to make the contamination problem undetectable. Sharma & Sharma (1986), working with lentils, and Pimentel (1990) working with maize, reported the harmful effect of radiation.

When the two concentrations (250 mg/50 mL vs. 750 mg/50 mL) were compared there was significance for GER and RAD, both in the presence (C3) and absence (C7) of radiation. This result shows that, either in absence or presence of radiation, the increase in concentration is harmful to germination and root growth.

The tendencies observed in the previous experiment indicated that a greater concentration of metronidazole (1,250 mg/50 mL) should be tested (Table 3). A significant result was obtained only for the CAU trait indicating little radiation effect (C1). The difference in result, when compared to C1 in Table 1, which also shows the effect of radiation, seems to be due to the lower percentage of germination and to the smaller development of the root and stem in the experiment involving the greater concentration of metronidazole (1,250 mg/50 mL). Thus these facts may have been confounded the radiation effect. The behavior of the means may be check by Table 4.

When a greater concentration of the substance was used (1,250 mg/50 mL), the effect of the metronidazole solution was found for all traits analyzed both in the presence (C2) and absence (C4) of radiation characterizing the action of metronidazole as a toxic substance and as a radiosensibilizer at a higher concentration. In addition, a synergistic effect of metronidazole with radiation was found for root length.

The effect of filtration on the solution was tested by contrasts 3 (with radiation) and 5 (without radiation). More significant effect of filtration was found at non-radiated treatments (C5), as in this case, the significance was confirmed for GER and CAU while for the irradiated treatments (C3) a significant effect was confirmed only for GER.

The radiosensibilizing effect of metronidazole has been described by Hall (1982) while investigating the effect of this substance combined with radiation on animal cells. Significant responses were found only for higher concentrations. The effects of this substance in plants, however, has not been reported. Santos (1993) displayed a similarity radiosensitizing effect in high concentration when seeds of maize were treated with mebendazole before irradiation. Olejniczak (1986, 1987) concluded that 70 Gy of gamma rays combined with sodium azide or MNUA (n-methyl-n-nitrosourea) markedly increased the number of chromosome aberrations in maize. Harley (1980), cited by Olejniczak (1986) has the opinion that an increase in the aberration number after the effect of combined mutagen is caused by inhibition of repair processes at the S phase or by inhibition of postreplication repairs. Cheng & Gao (1988) observed that a combined treatments (sodium azide and gamma rays) induced a higher mutation frequency with relatively less M1 injury, thus resulting in a higher mutation efficiency.

The preliminary test for cytological effect of metronidazole, performed under field conditions (M2 generation), reinforce the laboratory results, as the treatments with metronidazole show plants with semi-sterile pollen, probably due to the induction of structural chromosome aberrations. The treatment which involved only soaking in metronidazole solution showed one plant, among 25 analyzed, with semi-sterile pollen. The treatment which involved soaking and irradiation with 70 Gy showed four plants with semi-sterile pollen, among the 25 analyzed. All of these plants showed normal fenotypes. The appearance of a semi-sterile plant, without radiation may indicate the potential of metronidazole as an inducer of chromosome breaks.

Cytological studies should be carried out, to confirm the action of metronidazole as an inducer of alterations in the chromosome structure and also further studies should be done to check the effect of the substance as a radiosensibilizer in other plants.

CONCLUSIONS

1. At a high concentration (1,250 mg/50mL) metronidazole behaved as a radiosensibilizer.

2. Toxical effect of metronidazol was found at all concentrations studied.

3. The increase in metronidazol concentration is harmful to germination and root growth.

ACKNOWLEDGEMENTS

To the CNPq for financial support.

⁽²) Biology Department, Universidade Federal de Juiz de Fora, 36036-330, Juiz de Fora (State of Minas Gerais), Brazil.

⁽³) General Biology Department, Universidade Federal de Viçosa, 36571-000, Viçosa (State of Minas Gerais), Brazil.

• ANANTHASWAMY, H.N.; VAKIL, U.K. & SREENIVASAN, A. Biochemical and physiological changes in gamma-irradiated wheat during germination. Radiation Botany, Great Britain, 11(1):1-12, 1969.
• BOUVIER, L.; ZHANG, Y. X. & LESPINASE, Y. Two methods of haploidization in pear, Pyrus communis L.: grenhouse seedling selection and in situ parthenogenesis induced by irradiated pollen. Theoretical and Applied Genetics, Berlin, 87:229-232, 1993.
• CHENG, X.Y. & GAO, M.W. Biological and genetic effects of combined treatments of sodium azide, gamma rays and EMS in barley. Environmental and Experimental Botany, Great Britain, 28 (4):281-288, 1988.
• CUNY, F.; GROTTE, M.; DUMAS DE VAULX, R. & RIEU, A. Effects of gamma irradiation of pollen parthenogenetic haploid production in muskmelon (Cucumis melo L.). Environmental and Experimental Botany, Great Britain, 33 (2):301-312, 1993.
• DOYLE, G.G. A telocentric trisome and its potential use in the production of commercial hybrid corn using genic male sterility. Maize Genetics Cooperation Newsletter, Columbia, 46:142-146, 1972.
• GORLA, M.S.; VILLA, M. & OTTAVIANO, E. Pollen irradiation and gene transfer in maize. Maydica, Bergamo, 32:239-248, 1987.
• HALL, E.J. Radiobiology for the radiologist Hagerstown, Maryland, 1982. 512p.
• IQBAL, J. & ZAHUR, M.S. Effects of acute gamma irradiation and developmental stages on growth and yield of rice plants. Radiation Botany, Great Britain, 15:231-240, 1975.
• KOWYAMA, Y.; KAWASE, T. & YAMAGATA, H. Cell-cycle dependency of radiosensitivity and mutagenesis in fertilized egg cells of rice, Oryza sativa (L.). 2. X-ray sensitivity and mutation rate during a cell-cycle. Theoretical and Applied Genetics, Berlin, 68:297-303, 1984.
• MALUSZYNSKI, M.; AHLOOWALIA, B. S. & SIGUR-BJÖRNSSON, B. Application of in vivo and in vitro mutation techniques for crop improvement. Euphytica, Netherlands, 85:303-315, 1995.
• MUHANNA, S.; SOUVRÉ, A. & ALBERTINI, L. Effect of gamma rays on nuclear DNA and cellular RNA in meiocytes, microspores and tapetum of Nicotiana tabacum L. var. xanthi Dulieu. Cytologia, Tokyo, 56:107-115, 1991.
• OLEJNICZAK, J. Genetic variability in maize (Zea mays, L.) induced by mutagens. I. The effect of combined doses of N-methyl-N-nitrosourea, sodium azide and gamma rays. Genetica Polonica, Poznan, 27 (1/2):55-64, 1986.
• OLEJNICZAK, J. Genetic variability in maize (Zea mays, L.) induced by mutagens. II. Variability of morphological characteristics and yield structure components in the M2 and M3 plants of the maize line S-615 induced by combined treatments of sodium azide, N-methyl-N-nitrosourea and gamma rays. Genetica Polonica, Poznan, 28(1/2):43-51, 1987.
• PIMENTEL, M.C.G. Induçăo de aberraçőes cromossômicas estruturais em milho (Zea mays L) por radiaçăo gama Viçosa, 1990. 86p. Dissertaçăo (Mestrado em Genética e Melhoramento) - DBG, UFV, 1990.
• RAMAGE, R.T. Balanced tertiary trisomics for use in hybrid seed production. Crop Science, Madison, 5: 177-178, 1965.
• SANTOS, M.V.F.D.L. Resposta ŕ radiaçăo gama em sementes de milho (Zea mays L) sob a influęncia de agentes físicos e químicos Viçosa, 1993. 131p. Dissertaçăo (Mestrado em Genética e Melhoramento) - DBG, UFV, 1993.
• SHARMA, S.K. & SHARMA, B. Mutagen sensitivity and mutability in lentil. Theoretical and Applied Genetics, Berlin, 71: 820-825, 1986.
• SMITH, H.H. Radiation in the production of useful mutations. The Botanical Review, New York, 24(1):1-24, 1958.

⁽¹

) Financial support from CNPq. Received for publication in November 11, 1996 and approved in August 9, 1997.

⁽⁴

) PATTERSON, E.B. Proposed procedures for use of genetic male sterility in hybrid maize production. s.l., Illinois Corn Breeders School, 1971. (Not published.)

Publication Dates

• Publication in this collection
  16 June 1999
• Date of issue
  1997

History

• Received
  11 Nov 1996
• Accepted
  09 Aug 1997