Possible mechanisms of lethal and mutagenic action of formaldehyde

Mutation Research, 27 (1975) 123-126

© Elsevier Scientific Publishing Company, Amsterdam--Printed in the Netherlands

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Possible mechanisms of lethal and mutagenic action of formaldehyde The mutagenic properties of folanaldehyde have been known for a fairly long time. These properties are usually related to the capacity of formaldehyde to form adenine dimers through methylene bridgesZ, s. However, considering the mechanism of formaldehyde action on DNA in cells, we should remember that formaldehyde interacts, with considerably higher reactivity than with nitrogen bases, with amino groups of proteins and amino acids to form aminomethylol compounds s. Undoubtedly, these compounds are first formed when formaldehyde enters the cells. It is known that the monomethylol derivatives of amino acids can react with amide, guanidine, phenol, imidazole or indole groups3, 4 to form condensation products. We have previously reported that the products of reaction between CH~O and amino acids actively react with nucleotides and nucleic acidsZ°,zk In the present study we show th at treatment of wild strains of E. coli, and strains deficient in excision repair, with a product of reaction between CH20 and amino acid produces an inactivation of the cells as well as single-strand breaks in bacterial DNA. The breaks are successfully repaired in wild-type cells but remain unrepaiIed in bacteria deficient in DNA-polymerase I.

Materials and methods We used the isogenic strains of E. coli K 12 KS 16o thyA 324 argA- rha-lac y 14 strR and a mutant of this strain KS 66 polAI thyA 324 argA-rha-lac y 14 strR defective in DNA polymerase I. (We are grateful to G. B. Smirnov of the Gamaleya Institute for supplying the strains.) The procedure ot obtaining bacteria with ~Hlabelled DNA has been described in detail elsewhere 8. The formaldehyde solution was prepared as describedL and the concentration was determined by the iodometric procedure s. Glycine was purchased from Reanal (Hungary). Deoxycytidine-5'-phosphate was a Calbiochem (U.S.A.) preparation. The modification of dCMP by formaldehyde in the presence of glycine was checked spectrophotometrically. The composition of the solution in the control cuvettes differed from the reaction mixture only in the absence of the nucleotide. The experiments with the participation of formaldehyde and glycine were carried out using o.I M sodium phosphate buffer (pH 6.8). The variation of the pH of the buffer during the reaction between formaldehyde and amino acid was within the limits 6.5 and 6.8 for all the concentrations of amino acid and formaldehyde used. Results and discussion The reaction between nucleotides and formaldehyde conducted with an excess of glycine in the reaction mixture (with lespect to CH20) resulted in characteristic spectral changes, which differed significantly from those observed after treatment of nucleotides with formaldehyde only (Fig. I). Since the reaction occurred under conditions of incomplete modification of nucleotide 1° from the data in Fig. I one may assume that, for a given concentration oi formaldehyde (o.i M), a 3-fold molar excess of amino acid with respect to formaldehyde causes practically complete binding of aldehyde by amino acid and precludes the possibility of the usual formaldehyde reaction proceeding with nucleotide. This conclusion is in accordance in values with the literature for the equilibrium constant for the formation of monomethylolglycine

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during reaction of formaldehyde with glycine 5. Monomethylolglycine is the major product ot this reaction in the presence of glycine in the reaction medium at a surplus (with respect to CH~O) concentration and equilibrium constant, which allowed us to determine that the extent of formaldehyde binding (or monomethylolglycine formation) during interaction with glycine was about 60 1. mole -1 (ref. 5). Therefore, at a glycine concentration oi o.I M and a formaldehyde concentration of less than o.oi M binding of formaldehyde with amino acid will be practically complete. The experiments for studying the action of the product of formaldehyde reaction with glycine on the bacterial cells were conducted by using o.oo5-o.ol M solutions of formaldehyde with o.I M glycine. The results of these experiments were compared with the results of experiments in which bacterial cells were treated with formaldehyde only. As Fig. 2 shows, the survival of E. coli under the action of formaldehyde and of the product of the reaction between formaldehyde and amino acid are reduced to similar extents. Under these conditions, survival of the polA I mutant is significantly reduced as compared with the wild strain. 0.5 A

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Fig. I. Spectral changes of t h e dCMP solution caused b y t r e a t m e n t w i t h CH20 in the presence of glycine at various concentrations. Concentration of nucleotide a b o u t 5 " IO-~ M ; o.I M sodium p h o s p h a t e buffer (pH 7.0) ; o.i M formaldehyde. Beiore the s p e c t r a were recorded, the reaction m i x t u r e s were incubated for 18 h at 2o °. T h e composition of the solution in t h e control cuvettes differed from the reaction m i x t u r e only in the absence of nucleotide. I, The initial s p e c t r u m of the nucleotide solution in t h e absence of formaldehyde; 2, the s p e c t r u m of nucleotide after t r e a t m e n t w i t h CH20 in the absence of glycine; 3, CH20 w i t h o.i M glycine; 4, CH20 w i t h o.2 M glycine; 5, CH20 w i t h o.3 M glycine a n d w i t h higher concentrations. Fig. 2. Lethal effect of f o r m a l d e h y d e and of t h e p r o d u c t of the reaction of f o r m a l d e h y d e with glycine on E. coli strains KS 16o pol + (i) a n d KS 66 p o l A I (2). The cell culture g r o w n o v e r n i g h t was collected b y H U F S m e m b r a n e filters (Chemapol, Czechoslovakia), w a s h e d w i t h o.i M sodium p h o s p h a t e buffer (pH 7.o), a n d resuspended in this buffer. The cell suspension o b t a i n e d w a s divided into t w o portions, one of which was mixed w i t h the same v o l u m e of solution either of formaldehyde in t h e s a m e buffer or formaldehyde w i t h o.2 M glycine, and the second p o r t i o n used as a control was mixed with the same v o l u m e of buffer or buffer w i t h o.2 M a m i n o acid. T h e suspensions were incubated for 3o min a t 3 o°. The survival of the bacteria was determined b y the m e t h o d of macrocolonies. (©), F o r m a l d e h y d e ; ( + ) , formaldehyde w i t h glycine.

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We have studied the number of single-stranded breaks in the DNA of cells brought about b y the action of formaldehyde and of the product of the reaction between formaldehyde and amino acid on the wild and m u t a t e d strains (Fig. 3). The m u t a t e d strain had a large number of breaks in DNA after the action of CH,O and its products. Using the model experiments, we verified in our previous work the action on DNA of formaldehyde and of monomethylol derivatives of amino acids. The molecular weight of DNA sharply decreased under the action of the aminomethylol compounds and the acid-soluble products formed1°, 11. Formaldehyde itself is not capable of such action. So one m a y conclude that the single-strand breaks in the DNA of bacterial cells m a y be due to the action of the product of the reaction between formaldehyde and amino acids or they m a y be caused by the excision repair only. It is clear that, apart from the concrete mechanism of induction of the DNA lesions arising under the treatment of bacterial cells with formaldehyde or with monomethylolglycine and measured in our experiments as DNA single-strand breaks, these lesions (all or most of them) are effectively repaired in the wild-type cells under the participation of DNA polymerase I. This conclusion is based on the data of the present and other papers, and its discussion has been presented elsewhere 8. 16 14

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Fig. 3. C e n t r i f u g a t i o n of t h e D N A of s t r a i n s IKS 16o pol + a n d K A 66 polAI in a g r a d i e n t of a l k a l i n e sucrose. The cells l a b e l l e d in t h e l o g a r i t h m i c g r o w t h s t a g e b y [ a H ] t h y m i d i n e w e re r e s u s p e n d e d in o. i M p h o s p h a t e buffer a n d t h e s u s p e n s i o n o b t a i n e d w a s d i v i d e d i n t o t w o p o r t i o n s , one of w h i c h w a s m i x e d w i t h t h e s a m e v o l u m e e i t h e r of 2 • IO-3 M f o r m a l d e h y d e in o . i M p h o s p h a t e buffer (pH 7.o) or of f o r m a l d e h y d e s o l u t i o n w i t h 0.2 M glycine, a n d t h e s e c o n d p o r t i o n , u s e d as a control, w a s m i x e d w i t h t h e s a m e v o l u m e of buffer or buffer w i t h o.2 M glycine. The s u s p e n s i o n s wer e i n c u b a t e d for 6 m i n a t 37 °. The cell s u s p e n s i o n w a s c ol l e c t e d w i t h m e m b r a n e filters, res u s p e n d e d in an a p p r o p r i a t e v o l u m e of t h e s a l i n e s o l u t i o n a n d l y s e d b y l y s o z y m e a n d E D T A (ref. 8). P r o t o p l a s t s were l a y e r e d on an a l k a l i n e sucrose g r a d i e n t , 5 - 2 o % , in 0.2 N N a O H . A f t e r 3 ° m i n s t a n d i n g a t r o o m t e m p e r a t u r e , t h e t e s t t u b e s wer e c e n t r i f u g e d in a Spinco L5o c e n t r i f u g e (SVV-39 rotor, i o o rain, 3oooo r e v . / m i n ) . I, KS 16o pol + (KS 66 polAI) c o n t r o l ; 2, f o r m a l d e h y d e t r e a t m e n t of KS 16o pol + (in t h e a b s e n c e a n d in t h e prese nc e of glycine) ; 3, f o r m a l d e h y d e t r e a t m e n t of KS 66 polA I (in t h e a b s e n c e a n d in t h e p r e s e n c e of glycine).

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Thus, the d a t a o b t a i n e d here a n d in our previous study1°, n allow us to assume t h a t the action of formaldehyde on bacterial D N A (leading to lethality and, possibly, to mutagenesis) is n o t exerted b y formaldehyde itself b u t b y the products ot its reaction with a m i n o - c o n t a i n i n g c o m p o u n d s (amino acids, proteins etc.). The a s s u m p t i o n is based on the observation t h a t the action of formaldehyde a n d the effects of the m o n o m e t h y l o l derivatives of a m i n o acids on the c o m p o n e n t s of nucleic acids a n d D N A in vitro are quite differentl°, n, b u t the action on bacteria of both aldehyde a n d p r o d u c t of its reaction with a m i n o acid are similar: (i) b o t h are equally effective in producing cell i n a c t i v a t i o n ; (ii) t h e y induce the same q u a n t i t y of D N A lesions (measured as single-strand breaks) a n d (iii) i n d u c e d D N A lesions in the same bacterial strain are repaired (or unrepaired) with equal efficiency. I t should be remembered t h a t the c o m m o n pool of free a m i n o acids in bacterial cells is satisfactory 7 for effectively producing a m i n o m e t h y l o l c o m p o u n d s d u r i n g intracellular reaction with exogenous formaldehyde a n d t h a t the rates of i n t e r a c t i o n of formaldehyde with a m i n o acids a n d of the a m i n o m e t h y l o l c o m p o u n d s with nucleotides clearly exceed those of formaldehyde with nucleotides ~°.

Institute of Medical Radiology, Academy of Medical Sciences, Obninsk ( U . S . S . R . )

A . M. P O V E R E N N Y

Yu. A. SIOMIN A. S. SAENKO B. I. SINZINIS

I ALDERSON, T., Chemotherapy for a selective effect on mammalian tumour cells, Nature New

Biol., 244 (1973) 3-6. 2 BAYER, K., Analysis of Organic Compounds, Goskhimizdat, Moscow, 1953, P. 182, 3 FRAENKEL-CONRAT, H., AND H. OLCOTT, The reaction of formaldehyde with proteins, V. 4 5 6 7

Cross-linking between amino and primary amide or guanidyl groups, J. Am. Chem. Scc., 7° (1948) 2673-2684. FRAENKEL-CONRAT, H., and H. OLCOTT, Reaction of formaldehyde with proteins, VI. Crosslinking of amino groups with phenol, imidazole or indole groups, J. Biol. Chem., 174 (i948) 827-843. FRENCH., D., AND J. EDSALL, The reactions of formaldehyde with amino acids and protein, in M. L. ANSONAND J. T. EDSALL(Eds.), Advances in Protein Chemistry, Vol. 2, Academic Press, New York, 1945, pp. 278-335NISHIOKA, H., Lethal and mutagenic action of formaldehyde in Hcr + and Hcr- strains of Escherichia coli, Mutation Res., 17 (1973) 261-265. PIPERNO, J. R., AND D. L. OXENDER, Amino acid transport system in Escherichia coli K 12, J. Biol. Chem., 243 (1968) 5914-592o.

8 SAENKO, A. S., B. I. SINZlNIS AND G. n . SMIRNOV, On t h e ability of D N A p o l y m e r a s e - d e f i c i e n t

E. coli K 12 mutant polA I to rejoin single-stranded breaks of DNA induced by methyl methanesulfonate, Mol. Biol. (U.S.S.R.), 6 (1972) 655-663. 9 SIMONOV, V. ~7., N. I. RUABCHENKO AND A. M. POVERENNY, S t u d y of D N A d e n a t u r a t i o n w i t h

formaldehyde by differential spectrophotometry, 3Iol. Biol. (U.S.S.R.), i (1967) 297-3oi. io SIOMIN,YU. A., E. N. KOLOMIYTCEVAAND A. M. POVERENNY, The action of products of the reaction of formaldehyde and amino-acid on nucleotides and DNA, Mol. Biol. (U.S.S.R.), 8 (1974) 276-285 . I i SIOMIN, YU. A., V. V. SIMONOVAND A. M. POVERENNY, The reaction of formaldehyde with deoxynucleotides and DNA in the presence of amino acids and lysine-rich histone, Biochim. Biophys. Acta, 331 (1973) 27-32. Received May 6th, 1974