Document DvyrE18gOpRX1Dno7jKQo3zX4

3J So CO ?8osg0 PROG: i ,2 <.' AU - Lucier GW ; Lee IP ; Dixon rl AD v Natl."-Inst. Environ. H<--La'11 h,.; ic i . , NIH, Rese^r:c^ivjr.i.Ah*!>: .Par^ .-vC. TI -- Effects of environmental I aser, t s on mal e roFPoduction SI - CA/08S/032593D * ' *i .v:"C. SO - Testis' VOL 4.. 1977,577-604 - 'Jfr < if ' LA - ENG_ ( r AB - CBAW COPVrIQHI: CHEN ADS Pj review with many refs, of the effects of Pesticides, food additives ^nd contaminants, industrial chems., and air pollutants on male rV-prodn. ss 2 /c? USER: stop v JT.IME o: 03: 10 NLrt TIME 15=07: l>v PROG: nrinn-BYE 301 20 DT-.CONNECTED 00: 00: CO: 22 4: IS ' y-- -1\7 ' ?.i'C,'' >J. v . ' 'I'or'.v > v;.. w -:y,. R&S 025083 COMPLETE ARTICLE CAN BE FOUND IN TIER I B_K. -^Formaldehyde Acetaldehyde Pro pio na 1dehyde Butyraldehyde Acrolein Cro to na1dehyde Benzaldehyde For fu ra1 G1yoxal Maio naldehyde G1u ta ra 1dehyde Chloral Hydrate Hexamethylene Tetramine Ethyl Alcohol Vinyl Chloride K- 2574-dOO), K-2575K- 2 3 5 5- : K- 692K- 2576K- 693- | K- 715K -688- f' K-5489- [ 0167-5827K-20301 - K-2577- : K- 3057 - r GENETIC AND CYTOGENETICAL EFFECTS OF FORMALDEHYDE AND RELATED COMPOUNDS C. AUERBACH M. MOUTSCHEN-DAHMEN 5 and J MOUTSCHEN 3 1 Institute of Animal Genetics. Uniticrsity of Edinburgh, Hrstf Slams Bund, Etlmburgh, EII9 3JN (Great Britain) and 3 Labnratoirr dr Genetique, University de Liege. Hue Fnrgeur, 15. B-4000 Liege (Belgique) (Received February 9th, 1977) (Accepted March 2nd, 1977) Contents Historical outline..................................................................................... (A) The molecules........................................................................................... (B) Chemical reactions............................................................................ (a) Reactions with amino acids and proteins........................... fb) Reactions with nucleosides, nucleotides and nucleic avids (c) Reactions with nucleoproteins ................................................. (d) Formation of peroxides and pcroxidic radicals.................. (c) Precursor molecules . . . . . .............................................. (C) Oenetical and cylologic.il effects....................................................... (a) Formaldehyde .......................................................... *.................. (b) Other aldehydes ............................................................... ............... (c) Procurer molecules as potential mutagens..................... ... . (D) Applications and genetical hazards.................................................... Acknowledgements........................ ......................................................... References ........................... *.................................................................. 317 318 321 321 322 323 324 324 326 326 343 34 9 332 354 355 J3 B (/) o IN3 Ul O 00 -U Historical outline The cytological applications of formalin have been known since 1892 (ref. [179]). A lot of publications on its use as a fixative agent in mixtures for animal and plant cells have been written. More recently, it has been, suggested to replace formalin by other, less strong aldehydes especially glyoxal and glutaral- malrichyde on viral nucleoproteins has been amply exploited in the prepara tion of vaccines. Subsequently, it has been used as an inactivating agent of tobacco mosaic virus (TMV) [52,171] and transforming factor [188,189]. I i ; i v... . ;tih GcncLual research on formaldehyde started in 1LMG with Rapoporl's pubh cation on the production of sex-linked lethals in Drosophila larvae fed on medium containing formalin [135]. Until the middle fifties, this research wa<, followed up vigorously in several laboratories (section Ca) and was extended also to a few other aldehydes (section Cb). With the advent of the WatsonCrick era, interest shifted to chemical mutagens whose action at the molecular level could be more easily understood than that of formaldehyde. Towards the end of the fifties, research on formaldehyde mutagenesis had almost completely stopped. This has changed only recently. Over the last years, there has been renewed interest in the gcnetical effects of formaldehyde and other alde hydes. This has various reasons. First, the widespread use of these substances and their derivatives in industry and medicine (section D) calls urgently for an assessment of their potential hazards. Second, the reactions of formaldehyde with nucleotide bases and nucleic acids are now fairly well understood [-19] and find applications in studies on the molecular structure of DNA and RN'A. This should facilitate the formulation and testing of hypotheses on the molec ular nature of genetic damage by formaldehyde. Finally, the recent discovery that, in micro-organisms, formaldehyde-induced genetical effects are subject to excision, repair (section Ca) removes this substance from its isolated position and opens the way to analysis by means that have been successful in the study of other chemical mutagens. (A) The molecules The following list of compounds is not exhaustive. It deals only with sub stances that are wide-spread in the human environment and appear important from the point of view of genetic toxicity. Monofunctional saturated 'hcho ch2o H-C-H II O Methanal; formaldehyde; methylaldehyde; oxomethane; methylene oxide; oxymethylene; formic aldehyde. CHjCHO Ethanal; acetaldehyde; acetic aldehyde; ethylaldchyde. CH,CH,CHO Propanal; propionaldehyde; propylaldehyde; methylacetaldehyde. (CH3):CHCHO 2-Methylpropanal; isobutyraldehyde; fsobutyric aldehyde. (CHjLCCHO 2,2-Dimelhylpropanal; pivaldehyde; pivulaldehydc. SBOSg0 mmA'lrb -v.-./V*. '"vr/vatST.... m-.hw; IVW'MJBI 31 'i iicjciI;Choiicho 2,3-Dihydroxypropannl; idyceraldehyde; glyceric aldehyde; o /1-dihyriroxypropionaldehyde. CHjfCHjJjCHO Butanal; butyraldehydc; buLylaldehyde. CHj(CH,)3CHO Pentanal; valeraldehyde; valeric aldehyde; valeral. (CH,)5CHCH,CHO 2-MethylpenUinaJ; isovalderaldehydc. CHj(CH2)sCHO Heptanal; heptylaldehyde; heptaldehyde; aldehyde C-7. NHj(CH2)1NH(CH3):CHO Oxidized spermidine. CHjCHCOCjH^COCHO Kethoxal; fJethoxy-a-kefeibutylaldehyde. Monofunctional unsaturated CH3 = CHCHO 2-PropenaJ; acrolein; acrylic aldehyde; acrylaldehyde; acraldehyde. CHjCH = CHCHO TYa/is-2-butenal; crotonaJdehyde; crotonic aldehyde; 0-methylacrolein. CH, CHj 3,7-Dimcthyl-6-ortcnal; eitronellal; d-rhodinal (a mixture of stcreoisomeric aldehydes): CH2 = C(CH3)(CH2)3CH(CH3)CH2CHO (CH3)2C = CH(CH2)2CH(CH3)CH2CHO (CH3)2COH(CH3)3CH(CH3)CH2CHO Hydroxycitronellal (a saturated derivative). V cho CHj Cm, t-d 33 (/> 0 IO 01 o 00 0) T. ! UlfB lI . I t ' ir tn |lrt J( I * ? i \2* M i* 'PW >w> 320 3,7-DimethyJ-2,0-oct.'idif(iaJ; citral (from natural sources) - a mislurr of 2 geo. metric isomers : geranial anti nerai : (CHj)jC = C11(CH2)2CCH3 II UCCHO citral a = geranial (CHj)2C = CH(CH2)2CCH3 II OHCCH citral b = neraJ Monofunctional (cyclic compounds) Lo3-CHO Furfural; fural; 2-furaldehyde; pyromucic aldehyde; 2-furancarboxaldehyde hOCh,J JLchO o 5-(hydroxymethyl)-2-furfural; 5-(hydroxymethyl)-2-furfuraldehyde; 5-(hydroxymethyl)-2-furancarbonal; 5'hydroxymethyl-2-formy)furan; 11MF. CHO Benzenecarbonal; benzaldehyde; benzoic aldehyde; benzenecarboxaldehyde. CHO 6* o-Nitrobenzaldehyde. v- CH-------CHCHO 3-Phenylpropenal; cinnamaldehyde; cinnamic aldehyde; (3-phenylacrolein; cinnamal. Monofunctional with heterologous atoms CICHjCHO 2-Chloro-l-ethanal; chloroacetaldehyde; monochloroacetaldehyde. CljCCHO - HjO Chloral hydrate; ethanediol. trichloroacetaldehyde monohydrate; 2,2,2-trichloro-l.l- Bifunctional OHCCHO Ethanedial; glyoxal; biformyl; diformyl; oxalaldehytle. *% 025087 OHCCHjCHO Propanedial; malonaldehyde. it OHC(CHa)jCHO ^ pentanedial; glutaraldehyde; glutaric aldehyde. OHC(CH,),NH{CHJ)4NH(CHJ)iCHO Oxidized spermine. The natural or man-made distribution of alrlehydes in the human environment will be considered in section D. (B) Chemical reactions The reactions of formaldehyde with various components of increased com plexity and of biological importance have been exhaustively reviewed by Feld man [49] to whom we refer here. The reactions of other aldehydes have so far been much less investigated. Only those reactions likely to play a part in the production of genetical effects will be reviewed. Various hypotheses on the mechanism of formaldehyde mutagenesis have been based on these chemical findings; they will be discussed in section Ca. With formaldehyde, reactions with amino groups are certainly the most likely to occur in biological materials. These reactions can be summarized as follows; H R-NH2 + H-C it R.-N-CM2OM O II H O = C--N--H + H"C it O = C-N-CH2OH (I) (11) H HH I II r-n-ch2oh + r'-nh2 it r-n-ch2-n-r' + h2o (III) flla) Reactions with amino acids and proteins Reaction (I) occurs with the amino groups of amino acids and polypcptidic chains of proteins. The first step involves the formation of unstable methylol derivatives. The kinetics of the reactions with amino acids has been investigated a detail (for a review, see [54]). The second step of the reaction is as follows; IM R&S 025089 ; , I ni'i 'J*his slow step yields stable condensation products. I he condensation reaction of formaldehyde with proteins is proved, among other thirds, by the fact thoi after treatment of casein wiLh formaldehyde vapours, water is eliminated [121], The possibility of a formation of methylene bridges with proteins has been long debated. Under conditions close to physiological ones, the reaction of formaldehyde gives intermolecular crosslinks, but the opinions of the researchers differ somewhat in regard to the groups actually involved in the secondary reaction. In view of the important part played by enzymes in mutation and chromo some breakage, the reactions of formaldehyde with these molecules are of special significance for this review. This refers in particular to catalase which according to two investigations [172,175] is inhibited by formaldehyde while in a third [180] this could not be observed. The reaction of 2-chloroacetaldehyde with the sulfydryl group of glutathione and cysteine of rat liver has also been demonstrated (70,71). (Bb) Reactions with nucleosides, nucleotides and nucleic acids Reaction (II) occurs with nucleosides, nucleotides and nucleic acids. Reac tion (III) takes place only with amino purines. Reaction (II) can occur with the groups --CO--NH of pyrimidine and purine heterocycles.. As regards the biological action of formaldehyde, the reaction with NH groups in position 9 or 7 of the heterocycle ring of purines is especially sig nificant. Monomethylol derivatives involved in reaction (II) are only inter mediary labile products, which by secondary reaction give rise to stable methyl ene structures according to reaction (IV). In particular, the reactions of for maldehyde with compounds of the adenine series have received considerable attention. With AMP, a stable product methylene-bis-adenylic acid (V) is formed. In contrast, pyrimidine derivatives do not form stable condensation products with formaldehyde. -- CH,-- R R R ribo$c phosphate The kinetics of the reactions with purine has been carefully investigated (see [49]). The rate of the second step leading to methylene compounds is much slower than that of the first one: e.g. for adenosine, at room temperature and optimal conditions, more than a week is needed to get a negligible amount of the methylene compound, whereas the equilibrium ol the first step is reached within 70 h. The reactions of formaldehyde with DNA are less well known than those with nucleosides and nucleotides. Native double-stranded DNA does not react with formaldehyde. Treatments of DNA which destroy the hydrogen bonds allow the reaction to occur; after this reaction, DNA is not any more capable of renaturation. There is evidence that in a variety of conditions only unwound sites of DNA can react with formaldehyde and that these sites are not randomly distributed but occur in a rather regular fashion. The primary reaction of formaldehyde with both nucleic acids, resulting ^ monomethylol derivatives, occur1, as it r. tin- iw for smaller molecules. The valence is not so strong for the secondary ro.n tion. However, from the cxis.,.iiee of a fraction of formaldehyde firmly hound lo UNA it can he inferred tn.'it methylene bridges actually occur between purines, though there are still methods available to isolate these bridges from I)NA. Among other aldehydes, 2-chloroacctaldehyde is known to react with nucleosides in a specific way, forming fluorescent cyclic derivatives by acting pn the N-1 and N* nitrogens of adenosine and the N-3 and N4 nitrogens of cytiJi.ie 127,85,90], It can also react with DNA m single stranded regions. This p-action may be involved in the mutagenic effect of chloroacetaldehyde ,(100); section Cc). Binding of methylglyoxal to tRNA Mid DNA has been demonstrated (89). pirarbonyl compounds such as glyoxnl, ketho.xal and pyruvaldehyde interact with nucleic acids or their components in a different way from formaldehyde, possibly by two successive reactions with two carbonyl groups. In particular they do not form bridges between two bases (quoted in [-19)). Malonaldehyde has been reported to read with guanine and cytidine and to cross-link DNA [33]. Particularities of reactions with oxidized spermine will be discussed below. (Pc) Reactions with nucteoprotcins In reactions of aldehydes with nucleoproteins, steric factors should be taken into account, since they may accelerate these reactions. Special attention has Been given to nuclcohistones. It has been shown in pea buds that proteins closely associated with DNA are completely linked to it even by mild treat ments with formaldehyde [ 34 ], whereas under comparable conditions DNA itself is not cross-linked 153], All the data suggest that methylene bridges between nucleic acid and protein are responsible for formation of a complex with increased stability to heating (references in [49]). As models of the reaction with nucleoproteins, experiments in which various amino acids or lysine-rich histones were mixed with aldehydes during their reaction with nucleotides or DNA are of special interest. In the first experi ments [153], the following amino acids were mixed with deoxynucleotides or DNA during treatment with formaldehyde : L-histidine, DL-vnline, DL-alanine, DL-lysine and glycine. It was shown that these amino acids are involved in the reaction of formaldehyde with nucleic acid components, since the reaction products contained the amino acid residues. In addition, it was found that the rate of reaction of formaldehyde with DNA was enhanced in the presence of lysine-rich p. oteins [153). The kinetics of the reaction of glycine with nucleo tides, deoxynucleotides or DNA was investigated in further experiments with similar results [154]. These investigations demonstrated tha* niethylol deriva tives of the glycine or lysine-rich histones are first formed within a short time (several seconds) and interact secondarily with nucleotides or DNA. The sta bility of the end product depends on the nature of the amino acid and the nucleotides. The stability of products of the reactions with lysine seems greater than that of complexes with other amino acids [153], With glycine stability is greater for GMP than for other nucleotides. The reactions with DNA when amino acid or a lysine-rich fraction of histone arc added occur according to the ....... br Rl -------- *-%-- Ja>r ,' . 4( --------r :i2-i same funeral model. However, breaks appea/1 at a rather high rata | la.i.lal j j(} the sugar-phosphate moiety of the double helix. I li try produce an acid-soluble fraction of degraded molecules [15-1]. Some conclusions drawn from the experiments on formaldehyde interactions could be extended to other alde hydes, in particular in regard to the interactions with DNA [130]. Other researchers [186] are also of the opinion that the most dramatic action of formaldehyde is to induce DNA--protein cross-links some of which bind to the two strands of DNA. It should be remembered that the in vitro experiments monitored for purely chemical effects were intended to simulate in vivo conditions. Recent experiments with bacteria actually confirmed the conclusions drawn from in vitro ones [130]. (Bd) Formation of peroxides and peroxidic radicals It has long been shown that under several experimental conditions when oxidizing molecules are available, aldehydes can form reactive hydroxyalkyl peroxides and/or free radicals. The general reactions are as fodows. H R-C' r~c; + H' Free radical (VI) ,0-0' R-C' + 02 - R-C ^0 O free peroxy radical H o-o R--C + R-C ^o Nvo O-OH R--C. + R--C N"o ^o 1'eracid With H202 the reactions are: H R-C'' + II OO H " R-'CH(OH)--O--O "H ^O (VII) (VIII) (IX) R--CH--(OH)--OO-H + R-C^ A R--CH(OH)--O--O--CH(OH)R O In the particular case of formaldehyde: (X) H 2H-C^ + H-O-O-H;-' 110-CH,-O0-CH2-0H O dihydroxycimethyl peroxide (xD In vitro, a polymerization reaction of formaldehyde can occur, but the conden sation reaction is not energetic enough to explain biological effects [87], (Be) Precursor molecules In certain circumstances, some molecules can he considered as precursors of IF? wm jdehydcs. They m< hide llcxaincthvlenc tctranunc (urolropm) Condensation reactions of formaldehyde with ammonia can give rise to this compound. This reaction (Ml) is reversible, and at acidic pH's formaldehyde is slowly liberated. N \ (XII) fjhyl alcohol There has long been evidence that acetaldehyde is an intermediary product in ethyl alcohol metabolism. In vitro, it can be easily isolated as the first oxida tion product (XIII), and it is found in vivo in the blood during alcohol metab- ohsm. Acetaldehyde is very readily oxidized in vivo in acetic acid. ClU" -CHjOH _ <tu CH3--CHO (XIII) Vinyl chloride The muttaaggeenniic and carcinogenic effects of vinyl chloride have been attnbated to reeaaccttiioonn products formed in the liver by metabolic activation [134], One of these products is 2-chloroacetaldehyde. The most possible^routc is indi cated below (XIV). Other routes are considered unlikely in vitro (57). Cl--CH = CH* - Cl-CH-CHj ''O chloroethylene oxide (possible pathway) H *C1CH2--chloroacetaldchyde (XIV) Both metabolites appear in urine of man and rats exposed to vinyl chloride 165). Polyamincs Spermine and spermidine can give rise to aldehydes by oxidation, being transformed respectively into oxidized spermine (a dialdehyde) or oxidized spermidine (a monoaldehyde (Section A)). These are unstable substances which readily decompose by (3-elimination giving acrolein (XV). (XV) H Cll, -CH-C^ +CM3-NII (CH,).r-NH2 acrolein R&s 025092 iied 4t" ,/| *'L. K-,.- WJ There is certainly a potentl.il risk dm- to the appearance nf this aldi-hyd. (section Cb). Treatments of UNA with oxidized spermine result m coval.-nl cross-links of complementary chains which do not occur with oxidized spermi dine, suggesting that both carbonyl (troops are needed for cross links (23,2-1), The reaction of oxidized spermine is not specific for DNA since it can react with all bases containing amino groups and to a much lesser extent with thymine and uracil [24,4-1]. No firm conclusion can yet be drawn about the mechanism of reaction of these aldehydes, Pcroxidized polyunsturated fatty acids The oxidative decomposition of polyunsaturated fatty acids produces the three-carbon dialdehyde : malonaldehyde. The mutagenic effects of this sub stance will be dealt with in Section Cb. Dialky Ini trosamines Aldehydes are generated after oxidative monodealkylation by microsomal enzymes. They are thought to be rapidly further oxidized to yield CO) (for a survey, see [107]). ,CH,R O = N-N v CH2R nitrosamine ,CHjR ,CHjR , N--N N=N or XHR - 110 H + R--C_ HO (hypothetical) Triazcnes It has been shown that 1-phenyl 3,3-dimeLbyllriazenes wax dcalkylated to formaldehyde and probably monomethyltriazene by isolated microsoine frac tions from rat liver or to other organs, but only in presence of a NADPHgenerating system. The ultimate mutagen reacts with DNA like an alkylating agent [132], (C) Genetical and cytological effects (Ca) Formaldehyde K /l`l We start by discussing the findings on Drosophila that had been given for maldehyde in the food (FF), because these arc by far the most numerous and most fully analysed ones. Results obtained by exposure of Drosophila and other organisms to vapour or aqueous solutions of formaldehyde, and by com bination treatments between formaldehyde and other mutagens will be dealt with subsequently. Treatment of Drosophila with formaldehyde-food (FF) The first to test FF for mutagenic effects on Drosophila was Hapoport [135). He transferred eggs or young larvae (up to 48 h old) to food containing a "sublethal" admixture of formaldehyde. The emerging males were tested by the C1B method and yielded about 6% lethals, in some experiments more. Kap lan repeated these experiments with similar results [73], He transferred larvae R&s 025093 *>**.', , H. and strange enough, this denaturation process is prevented by some cations such as sodium. It is difficult to say to what extent these in vitro findings can be extrapolated to in vivo situations. A clear picture of the relative importance of the different mechanisms of action does not yet emerge. A possible way to tackle this question would be to look for correlations between the effects of the drug on cellular processes and its well-known anaesthetic action. (Cc) Precursor molecules as potential mutagens Hexamethylene tetrarnine (urotropin) t r _ This substance, its salts and other substances of the same type as ampho- tropin, helmitol and anotropin were suggested as potential mutagens. Rapoport [135,136] states that these substances can produce mutations in Drosophila when mixed with the larval medium at much higher concentrations than those used in therapy. Ethyl alcohol k' 1 ~ - '*'? Ethyl alcohol by itself has a chromosome breaking action in Vicia faba [104] and induces specific X-chromosome breakage in the grasshopper Phloeoba antennata but not in another species Oxya uelox [98]. Since there is a great variety of response between organisms, possibly due to different metab olic pathways, it is not yet possible to evaluate which effect can be attributed to acetaldehyde. Vinyl chloride k'-f ' H The mutagenic and carcinogenic effects of vinyl chloride have been reviewed [29], Chloroethanol can be considered as an intermediary step leading to chloroacetaldehyde. The toxicity of chloroethanol in the rat was attributed to chloroacetaldehyde [71], The mutagenic effects of one of its metabolites, chloroacetaldehyde, are described in section Cb. Polyamines As mentioned before (section Cb), the aldehydes produced by oxidation of spermine and spermidine are toxic for a certain number of organisms. Since the decomposition of these products can give rise to acrolein by (1-elimination, this creates a potential hazard. However, without oxidation, polyamines are not likely to be mutagenic. In fact, spermine has been found to be an antirnutagen [72]. Peroxidized polyunsaturated fatty acids These substances hove not yet been tested as such for genetical effects. The effects of one of its metabolites, malonaldehyde, are described in Section Cb. Dtalkylnitrosamines and triazenes As compared with the strong mutagenicity of the activated metabolites obtained in the reactions with microsomes it is unlikely that the amount of aldehydes produced can induce a significant part of the mutations observed [107,132 ].