Mutation Research, 264 (1991) 135-137 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0165-7992/91/$03.50 ADONIS 0165799291000958
135
MUTLET 0543
Mutagenic effects of aluminium J.C. Octive, M. Wood and A.C. Johnson Department of Soil Science, The University of Reading, London Road, Reading RG I 5A Q (Great Britain)
(Received 5 March 1991) (Revision received 2 July 1991) (Accepted 12 July 1991)
Keywords. Aluminium; Alzheimer'sdisease; DNA, binding of AI to
Aluminium, the third most c o m m o n element in the earth's crust, is ubiquitous in the natural and domestic environment, and has been implicated in fish and tree death as a result of acid rain, and in the disabling h u m a n neural condition known as Alzheimer's disease (Sigel and Sigel, 1988). A1 is known to bind to D N A of m a m m a l s (Karlick et al., 1980), plants (Morimura and Matsumoto, 1978) and bacteria (Johnson and Wood, 1990). During studies on the mechanism of A1 toxicity to the c o m m o n soil bacterium Rhizobium, we have found circumstantial evidence that A1 is mutagenic: (i) The effects of A1 on cell viability and morphology (elongated cells) were similar to those of mitomycin C (unpublished results), a known mutagen (Moseley and Copland, 1978). (ii) A1 was found to penetrate the cell envelope and bind to D N A in R h i z o b i u m leguminosarum biovar trifolii strain RDG 2002. Despite causing a reduction in viability, AI stimulated D N A synthesis in this strain (Johnson and Wood, 1990); in Escherichia coli such a response to D N A damage is Correspondence: Dr. J.C. Octive, Department of Soil Science, The University of Reading, London Road, Reading RG1 5AQ (Great Britain).
associated with the mutagenic SOS-type repair response (Walker, 1985). We report here more conclusive evidence for mutagenic effects of A1 based on changes in antibiotic resistance following exposure of this Rhizobium strain (and one other) to A1. Mitomycin C was also used, as a positive control. Materials and methods
The methods used are based on those of Cunningham (1979). R h i z o b i u m leguminosarum biovar trifolii strain R D G 2002 (isolated in our laboratory), and R. loti strain N Z P 2037 (obtained from DSIR, Palmerston North, New Zealand) were maintained at 5°C on yeast extract mannitol (YEM) agar incorporating 3 g CaCO3 1-1 (Vincent, 1970). These strains have been used in previous studies (Johnson and Wood, 1990) and are representative of their species. The number of viable cells was measured using serial dilutions and YEM agar. Strains were exposed to A1 or mitomycin C in a defined arabinose-galactose-glutamate liquid medium (Wood and Cooper, 1988). Strains RDG 2002 and N Z P 2037 were grown in liquid defined medium at p H 5.5 on a rotary shaker
136
TABLE 1 SURVIVAL OF RHIZOBIUM STRAINS DURING EXPOSURE TO 50 /zM ALUMINIUM OR 10 #g ml ~ MITOMYCIN C Strain
Treatment
Log (number of c.f.u, ml- ~) Initial
After 18 h
RDG RDG NZP NZP
Mitomycin C AI Mitomycin C A1
7.5 7.9 8.0 8.0
2.9 8.0 5.7 8.0
2002 2002 2037 2037
(75 r.p.m.) at 25°C until late exponential phase (approximately 108 cfu ml-~) then A1 (50 #M as AIK(SO4)z) or mitomycin C (10 #g m l - t ) were added and the cultures incubated for a further 18 h. Viable counts were made prior to the addition of the A1 or mitomycin C and 18 h after their addition. Cells were then centrifuged for 30 rain at 2000 g and washed twice with deionised distilled water. Pellets were resuspended in liquid defined medium and incubated for 48 h as above before being tested for rifampicin resistance on YEM agar plates containing 0, 25, 50 or 75 p.p.m, rifampicin and incubated at 25°C for a period of 3-14 days. All treatments were done in duplicate and controls (without AI or mitomycin C) were included. Results and discussion
In this experiment 50 #M AI was not toxic to either strain (Table 1), however, 10 #g ml -~ mitomycin C was toxic to part of the population. NZP 2037 has been shown previously to be more tolerant to AI than RDG 2002 (Johnson and Wood, 1990) although in this experiment in which the cells were exposed to AI in late exponential phase there was no difference. Previous studies have shown that strains are more sensitive to AI during earlyand mid-exponential phases of growth than in latestationary stage (Wood and Cooper, 1988). The data in Table 2 show that the rate of spontaneous mutation for rifampicin resistance in both strains was < 10 -58, but after exposure to A1 the incidence of rifampicin resistance was much higher (10 -2.8 for R D G 2002 and 10 5.0 for N Z P 2037).
The mutation rate for rifampicin resistance caused by mitomycin C was lower than that caused by AI (Table 1); there was an increase in the incidence of rifampicin resistance for RDG 2002, but not for NZP 2037. These data show several similarities between the response by Rhizobium to AI and to mitomycin C, which is a known mutagen (Moseley and Copland, 1978). Greater tolerance to A1 in N Z P 2037 (as previously shown by Johnson and Wood, 1990) was associated with greater tolerance to mitomycin C (Table 1), and a lower incidence of mutation in response to AI was associated with a lower mutation rate in response to mitomycin C (Table 2). This was predicted from the mechanism of AI toxicity proposed by Johnson and Wood (1990) in which the binding of AI to DNA in strain RDG 2002 caused an increase in DNA synthesis (as shown by thymidine incorporation). This also occurs with the SOS repair response in E. coli (Walker, 1985) and is also mutagenic. No increase in D N A synthesis in response to AI damage was observed for N Z P 2037 (Johnson and Wood, 1990), and a lower incidence of mutation in response to AI was observed here. The data show that an error-prone repair system operates in Rhizobium in response to damage to D N A caused by mitomycin C and AI. The lower incidence of mutation by the more Al-tolerant strain in response to A1 suggests that the tolerance
TABLE 2 RIFAMPICIN RESISTANCE OF RHIZOBIUM STRAINS FOLLOWING EXPOSURE TO 50/~M ALUMINIUM OR 10 tzgml ~ M I T O M Y C I N C Strain
RDG RDG RDG NZP NZP NZP
2002 2002 2002 2037 2037 2037
Treatment
Control Mitomycin C AI Control Mitomycin C AI
Growth of cells (log number of c.f.u, m l - I ) on YEM agar containing No. rif.
25 /zg ml-t rif.
8.08 7.34 8.26 8.11 8.14 8.38
135
MUTLET 0543
Mutagenic effects of aluminium J.C. Octive, M. Wood and A.C. Johnson Department of Soil Science, The University of Reading, London Road, Reading RG I 5A Q (Great Britain)
(Received 5 March 1991) (Revision received 2 July 1991) (Accepted 12 July 1991)
Keywords. Aluminium; Alzheimer'sdisease; DNA, binding of AI to
Aluminium, the third most c o m m o n element in the earth's crust, is ubiquitous in the natural and domestic environment, and has been implicated in fish and tree death as a result of acid rain, and in the disabling h u m a n neural condition known as Alzheimer's disease (Sigel and Sigel, 1988). A1 is known to bind to D N A of m a m m a l s (Karlick et al., 1980), plants (Morimura and Matsumoto, 1978) and bacteria (Johnson and Wood, 1990). During studies on the mechanism of A1 toxicity to the c o m m o n soil bacterium Rhizobium, we have found circumstantial evidence that A1 is mutagenic: (i) The effects of A1 on cell viability and morphology (elongated cells) were similar to those of mitomycin C (unpublished results), a known mutagen (Moseley and Copland, 1978). (ii) A1 was found to penetrate the cell envelope and bind to D N A in R h i z o b i u m leguminosarum biovar trifolii strain RDG 2002. Despite causing a reduction in viability, AI stimulated D N A synthesis in this strain (Johnson and Wood, 1990); in Escherichia coli such a response to D N A damage is Correspondence: Dr. J.C. Octive, Department of Soil Science, The University of Reading, London Road, Reading RG1 5AQ (Great Britain).
associated with the mutagenic SOS-type repair response (Walker, 1985). We report here more conclusive evidence for mutagenic effects of A1 based on changes in antibiotic resistance following exposure of this Rhizobium strain (and one other) to A1. Mitomycin C was also used, as a positive control. Materials and methods
The methods used are based on those of Cunningham (1979). R h i z o b i u m leguminosarum biovar trifolii strain R D G 2002 (isolated in our laboratory), and R. loti strain N Z P 2037 (obtained from DSIR, Palmerston North, New Zealand) were maintained at 5°C on yeast extract mannitol (YEM) agar incorporating 3 g CaCO3 1-1 (Vincent, 1970). These strains have been used in previous studies (Johnson and Wood, 1990) and are representative of their species. The number of viable cells was measured using serial dilutions and YEM agar. Strains were exposed to A1 or mitomycin C in a defined arabinose-galactose-glutamate liquid medium (Wood and Cooper, 1988). Strains RDG 2002 and N Z P 2037 were grown in liquid defined medium at p H 5.5 on a rotary shaker
136
TABLE 1 SURVIVAL OF RHIZOBIUM STRAINS DURING EXPOSURE TO 50 /zM ALUMINIUM OR 10 #g ml ~ MITOMYCIN C Strain
Treatment
Log (number of c.f.u, ml- ~) Initial
After 18 h
RDG RDG NZP NZP
Mitomycin C AI Mitomycin C A1
7.5 7.9 8.0 8.0
2.9 8.0 5.7 8.0
2002 2002 2037 2037
(75 r.p.m.) at 25°C until late exponential phase (approximately 108 cfu ml-~) then A1 (50 #M as AIK(SO4)z) or mitomycin C (10 #g m l - t ) were added and the cultures incubated for a further 18 h. Viable counts were made prior to the addition of the A1 or mitomycin C and 18 h after their addition. Cells were then centrifuged for 30 rain at 2000 g and washed twice with deionised distilled water. Pellets were resuspended in liquid defined medium and incubated for 48 h as above before being tested for rifampicin resistance on YEM agar plates containing 0, 25, 50 or 75 p.p.m, rifampicin and incubated at 25°C for a period of 3-14 days. All treatments were done in duplicate and controls (without AI or mitomycin C) were included. Results and discussion
In this experiment 50 #M AI was not toxic to either strain (Table 1), however, 10 #g ml -~ mitomycin C was toxic to part of the population. NZP 2037 has been shown previously to be more tolerant to AI than RDG 2002 (Johnson and Wood, 1990) although in this experiment in which the cells were exposed to AI in late exponential phase there was no difference. Previous studies have shown that strains are more sensitive to AI during earlyand mid-exponential phases of growth than in latestationary stage (Wood and Cooper, 1988). The data in Table 2 show that the rate of spontaneous mutation for rifampicin resistance in both strains was < 10 -58, but after exposure to A1 the incidence of rifampicin resistance was much higher (10 -2.8 for R D G 2002 and 10 5.0 for N Z P 2037).
The mutation rate for rifampicin resistance caused by mitomycin C was lower than that caused by AI (Table 1); there was an increase in the incidence of rifampicin resistance for RDG 2002, but not for NZP 2037. These data show several similarities between the response by Rhizobium to AI and to mitomycin C, which is a known mutagen (Moseley and Copland, 1978). Greater tolerance to A1 in N Z P 2037 (as previously shown by Johnson and Wood, 1990) was associated with greater tolerance to mitomycin C (Table 1), and a lower incidence of mutation in response to AI was associated with a lower mutation rate in response to mitomycin C (Table 2). This was predicted from the mechanism of AI toxicity proposed by Johnson and Wood (1990) in which the binding of AI to DNA in strain RDG 2002 caused an increase in DNA synthesis (as shown by thymidine incorporation). This also occurs with the SOS repair response in E. coli (Walker, 1985) and is also mutagenic. No increase in D N A synthesis in response to AI damage was observed for N Z P 2037 (Johnson and Wood, 1990), and a lower incidence of mutation in response to AI was observed here. The data show that an error-prone repair system operates in Rhizobium in response to damage to D N A caused by mitomycin C and AI. The lower incidence of mutation by the more Al-tolerant strain in response to A1 suggests that the tolerance
TABLE 2 RIFAMPICIN RESISTANCE OF RHIZOBIUM STRAINS FOLLOWING EXPOSURE TO 50/~M ALUMINIUM OR 10 tzgml ~ M I T O M Y C I N C Strain
RDG RDG RDG NZP NZP NZP
2002 2002 2002 2037 2037 2037
Treatment
Control Mitomycin C AI Control Mitomycin C AI
Growth of cells (log number of c.f.u, m l - I ) on YEM agar containing No. rif.
25 /zg ml-t rif.
8.08 7.34 8.26 8.11 8.14 8.38
