Toxicological evaluation in rats and mice of the ingestion of a cheese made from milk with added formaldehyde

Fd Chem. Toxic. Vol. 21, No. 3. pp. 313 to 317. 1983 Printed in Great Britain. All rights reserved

0278-6915,,83,030313-05503.00/0 Copyright © 1983 Pergamon Press Ltd

TOXICOLOGICAL EVALUATION IN RATS A N D MICE OF THE INGESTION OF A CHEESE MADE FROM MILK WITH A D D E D F O R M A L D E H Y D E C. L. GALLI, C. RAGUSA, P. RESMINI* and M. MARINOVICH Laboratory o1"Toxicolo:ty, Institute ~!1Pharmacolo~ty and Pharmaco~lnosy, Unit'ersity of Milan. Via Andrea del Sarto, 21-20129 Milan

and *lstituto di lmlustrie. A:lrarie, Uni*'ersity q/Milan, Italy (Receil'ed 2 Au:lust 19~23

Abslract Male Swiss albino mice (CD-I) and male Sprague Dawley rats were given single oral doses of 0.5 g (4.0 ttCi) and 2.2 g (18 l~Ci) '~C-labelled grana cheese, respectively. The cheese was made by the normal process but using milk with added [l"*C]formaldehyde. The plasma and tissue kinetics of the radiolabelled cheese were studied by monitoring the decay of radioactivity in the plasma, gastrointestinal tract, liver, kidney, lung, testes, spleen, brain, muscle and adipose tissue. The faeces and urine of animals placed in individual metabolism cages were collected between 4 and 64 hr after dosing for rats and between 2 hr and 12 days for mice. Within 32 hr of administration 63 67°. of the radioactivity had been excreted in the faeces and urine and 24 28". of the radioactivity had been exhaled as ~4CO2, in both species. Maximum concentrations, corresponding to 0.07",, and 0.3". of the dose per ml of blood were reached respectively within 8 hr for rats and 2 hr for mice. The toxicokinetic profile appears to be similar in mice and rats because of the similarity of the half-lives of the climination phase, 27.8 and 26.4hr respectively, and suggests that accumulation of the ~'~C-activity does not occur in any of the tissues of either species. The low levels of radioactivity still present 32 hr after the administration of >*C-grana cheese are probably due to the residues of labelled fractions of milk protein not completely metabolized.

INTRODtCTION Formaldehyde is a normal cellular metabolite and participates in the one-carbon pool. At present, formaldehyde is a significant commercial chemical with a worldwide consumption of about 5 million tons annually. Sources of exposure of the public to formaldehyde include cigarette smoke, motor-vehicle exhausts, photochemical smog, incinerators, and release from urea-formaldehyde products. The epidemiological evidence concerning the carcinogenicity of FA to man, provides no proof of a causal relationship between previous exposure and the occurrence of malignant neoplasms (Marsh, 1982; Walrath & Fraumeni, 1982; Wong, 1982). However, recent evidence has emerged that links exposure to formaldehyde with nasal tumours in rats and mice (Kerns, 1980: Swenberg, Kerns, Mitchell et al. 1980). Formaldehyde reacts with proteins (French & Edsall, 1945) and nucleic acids (Chaw, Crane, Lange & Shapiro, 1980; Collins & Guild, 1968- Feldman, 1973; Lewin, 1966) and it has been reported to induce D N A protein crosslinks (Thomas, 19763 both in mouse LI 210 cells (Ross & Shipley, 19803 and V 79 cells (Swenberg, Gross, Martin & Popp, 1982l. A recent study on human lung cells in t'itro indicated that t~*c from "~C-labelled formaldehyde is incorporated preferentially into RNA rather than DNA or nuclear proteins (Pruett, Scheuenstuhl, Michaeli & Nevo, 1980). D N A damage has been observed in bacteria (Rosenkranz & Leifer, 19801, yeasts (Chanet & von Borstel, 1979) and mammalian cells (Martin, 313

McDermid & Garner, 1978: Obe & Beek, 1979) exposed to formaldehyde. The genetic effect of formaldehyde in Drosophila melanogaster depends on the mode of administration. Formaldehyde added to the food resulted in a strong mutagenic effect which was restricted to early larval spermatocytes (Auerbach, 1967: Auerbach, Moutschen-Dahmen & Moutschen, 1977). No dominant lethals were induced in Swiss (ICR/Ha) mice injected ip with 16-40mg formaldehyde/kg body weight (Epstein, Arnold, Andrea et al. 19723. Formaldehyde is absorbed after inhalation or ingestion in dogs (Egle, 1972; Malorny, Rietbrock & Schneider, 1965) and, to a lesser extent, when applied to the skin of guinea-pigs (Usdin & Arnold, 19793. Whole-body autoradiography of mice following iv injections of [l'~C]formaldehyde showed that it was localized in the liver and, to a lesser degree, in the kidney (Johansson & Tj~ilve, 1978). After sc (du Vigneaud, Verly & Wilson, 19503 or ip (Neely, 1964) injection of t4C-labelled formaldehyde in rats 81 and 82!',, of the radioactivity respectively was recovered as respired 14CO z. Reproduction studies, including teratogenicity studies, in dogs fed diets containing 125 or 375 ppm formaldehyde or 600 or 1250ppm of hexamethylenetetramine {HMT), which gradually decomposes to yield formaldehyde and ammonia in acid solution or in presence of proteins, did not reveal any compoundrelated adverse effect (Hurni & Ohder, 19733. No evidence of carcinogenicity was found when 0.5~'o and

('. k. GALLI eta/.

314

1% H M T were given in the drinking water for 60 and 104wk to rats and mice respectively (Della Porta, Colnaghi & Parmiani, 1968). Neither were any carcinogenic effects observed when rats were given 1% H M T in the drinking water for three successive generations (Della Porta, Cabral & Parmiani, 1970). The use of formaldehyde as an antimicrobial agent in food (such as milk) has caused concern, mainly because of the lack of data on the toxicity of formaldehyde administered by the oral route and because of the potential hazard caused by the stable reaction compounds between fnrmaldehyde and food proteins. The aim of the present work was to study the absorption, fate and excretion of the complexes between ~'~C-labelled formaldehyde and milk proteins in mice and rats and to discuss their toxicological significance. EXPERIMENTAL

Materials and animals. Unlabelled formaldehyde (Carlo Erba, Milano, Italy) and 14C-labelled formaldehyde (Amersham International pie, Amersham, Bucks, UK) with a specific activity of 17.6 mCi/mmol were added to milk to obtain a final concentration of 35 40 ppm. This is the concentration used in the norreal production of grana cheese to achieve a nonselective bacteriostatic action on the milk microflora so that the milk can be partially skimmed by static separation in trays without the alterations in microflora that often occur at this stage leading to failure in the cheese-making process. Radioactive grana cheese was made following the usual procedures used in grana cheese making (Resmini, Saracchi & Motti, 1980b). Male Sprague--Dawley rats (Charles River Inc. Calco. Italy) weighing 150 200g and Swiss albino mice CD-I (Charles River Inc.) 25 30g body weight were used in the two experiments. The constituents of the animals' normal diet were: wheat maize flour, soyabean flour, wheat bran, meat flour, fish meal, alfalfa flour, powdered milk whey, grist, non-fat milk powder, C a H P O 4 NaC1, vitamin mix, and BHT (4 mg). Experimental desiqn and conduct. Animals trained to eat during l hr which was followed by 12hr of fasting were placed individually in all-glass metabolism chambers equipped for the collection of faeces and urine (Disa, Milano, Italy) and given 1'*C-labelled cheese, in doses of 2.2g (18tLCi; rats) and 0.5g (4.0 llCi; mice). Animals fed with unlabelled cheese were used as controls. Groups of rats were killed 4, 8. 16, 32 and 64 hr after the end of food consumption, and mice were killed after 2, 4, 8. 16, 32, 64 and 96 hr and 8 and 12 days. Blood, liver, gastro-intestinal tract, kidneys, lungs, spleen, testes, brain, muscle and adipose tissue were removed. Urine and faeces were collected from the metabolic chambers. In all cases the biological samples were frozen on dry ice immediately after removal and kept at - 2 0 ~ C until used for radioactivity measurement. Determination of radioacticity. After homogenization, duplicate samples of approximately 10 20 mg of all tissues were solubilized in 1.0 ml Lumasolve (Lumac System AG, Basel, Switzerlandl at 5OC for 30rain. Lipoluma (5ml, Lumac System AGI was added to all vials as scintillation liquid. Blood (50/d

aliquotst was added to 0.5 ml of a Lumasolve isopropanol solution (l:2vx.} and shaken for 60rain al 50 C, after which 0.5 ml 35",, (WV) H 2 0 2 was added dropwise to each vial, the samples werc kept for 30rain at room temperature and then 101nl Lumagel 0.Sy-HC1 (9:1. v'v) were added its scintillation liquid. To 501tl of the solution of urine and faeces 0.5ml Lumasolve isopropanol 11:2. v.v) was added and the samples were digested overnight at 50 C with shaking. Cooled samples were bleached with 0.5 ml (v/v) H 2 0 2. and 15ml Lumagel 0.5N-HCI (9:1.'~ v) were added before cotmting faeces and twine. Levels of >*C-activity in the samples were determined with a Packard TriCarb 3255 scintillation counter. Collection and determblation (!II4('0,. The air in the chamber was pulled through a train consisting of three glass traps containing 150ml of cellosolve ICarlo Erba, Milano. Italy)in the [irst trap and cellosolve ethanolamine 175:25. v v l in the following tx~o traps to ensure complete absorption of the released CO2. Air was drawn through this system at rates of 35 and 50 litre'hr for mice and rats respectively by a vacuum pmnp. The traps were changed at 8- to 16-hr interwds. To analyse the CO, content, to sltmples from each trap 11.0 roll, 5 ml of methanol and 10 ml of Lipoluma were added and the radioactivity counted in a Packard TriCarb 3255 scintillation counter. The efficiency of the counter was determined for each sample using extenal standard channel ratios and referring to appropriate quenching curves. Tissues of control animals were processed in the same manner and the counts generated were considered to be the background radioactivity levels. Statistical amllysis. Statistical signilicance was determined by two-tailed Student's I test or b \ the Dunnett's analysis. RESI LTS After a single oral administration of t4C-grana cheese the animals showed no sign of a b n o r m a l hchaviour or appearance. N o significant difference was

lbund between treated and control animals in body or organ weights or in food consumption. In mice the highest concentrations of radioactivity in the liver, kidney, adipose tissue, spleen, testes, brain and muscle occurred 4 h r after the administration (Table 1). The radioactivity profile of the blood indicated that the maximum concentration corresponding to 0.3% of the dose was reached within 2 hr (Table 1). In rats peak radioactivity concentrations in the tissues mainly occurred 16hr after food consumption. In blood the highest concentrations of ~4C-activity, equivalent to a value of 0.08",, of the dose. x~cre present after 8 hr (Table lt. The rate of disappearance of the radioactivity from tissues and blood suggests that accumulation of the product obtained from [~'~C]formaldehyde and milk proteins and.or its metabolites does not occur in either species. The half lives calculated from the regression line of the fl phase (elimination phase) in blood were found to he 27.8 hr and 26.4 hr for the mouse and rat respectively. After 96 hr in all the mouse tissues the radioactiviD conccntration/g tissue was lower than 0.5"ii of the administered dose and no radioactivity was detectable after days [Table 1).

3 4 10 12 4 4 7 3 3

3 3 3 3 3

2hr 4hr 8hr 16hr 32hr 64hr 96hr 8 days 12 days

4hr 8hr 16hr 32hr 64hr

209.0 171.5 174.3 8.9 1.4

+- 18.2 _+ 7.3 + 4.3 + 3.3 _+ 0.1

282.7 + 16.4 175.6 _+ 11.1 143.7+ 1 1 . 5 22.5 _+ 11.4 1.6 + OAt 0.8 _+ 0.05 0.6 + 0.05 ND ND

Gastrointestinal tract

13.4 11.2 18.0 9.8 7.9

_+ 0.9 +- 0.7 -+ 2.1 +- 0.9 + 1.6

10.2 _+ 0.5 11.6 + 1.7 8.4_+0.8 6.8 + 0.6 6.4 +_ 1.7 3.0 + 0.2 2.2 -+ 0.09 ND ND

Liver

52.0 47.9 41.3 7.6 3.6

+ 1.1 _+ 1.8 -+ 1.3 + 0.4 + 0.7

22.9 + 3.9 39.0 _+ 4.2 38.5 + 5.1 15.9 +- 1.7 10.8 + 0.7+ 6.1 _+ 0.6 3.8 + 0.7 ND ND

Kidney

7.4 7.0 8.6 2.9 2.0

+ + + + +

0.7 3.0 1.4 0.2 0.5

7.5+ 7.9 + 0.8 4.8_+0.7 2.8 + 0.6 1.4 _+ 0.09 1.5 + 0.2 1.6 _+ 0.2 ND ND

Adipose tissue

Rats

Mice

2.4 2.9 2.6 1.3 0.7

_+ 0.l + 0.3 _+ 0.4 + 0.05 + 0.2

2.3 + 0.01 2.1 _+ 0.1 1.7+0.08"t" 0.9 _+ 0.1+ 0.6 + 0.09 0.5 _+ 0.04 0.8 + 0.1"I" ND ND

Blood*

3.7 5.6 7.2 4.0 2.5

_+ 0.3 + 0.3 + 0.3 + 0.1 _+ 0.3

3.4 + 0.6 3.9 + 0.7 2.6+0.2 1.7 + 0.2 0.8 _+ 0.2 0.7 _+ 0.2 0.8 _+ 0.06 ND ND

Spleen

3.9 4.0 4.6 2.7 2.0

+ 0.09 _+ 0.3 -+ 0.3 _+ 0.2 + 0.2

3.0 -+ 0.3 2.9 + 0.2 2.5+_0.2 1.9 + 0.2+ 1.4 _+ 0.2 0.9 + 0.07 0.8 _+ 0.05 ND ND

Lung

2.0 2.4 3.3 1.6 1.4

_+ 0.2 _+ 0.2 -+ 0.3 _+_0.05 + 0.1

1.7 -+ 0.1 1.8 + 0.2 1.4_+0.1 1.0 + 0.1 0.8 -!-_0.4 0.6 _+ 0.05 0.6 _+ 0.08 ND ND

Testes

1.9 2.1 3.1 1.5 0.9

+ 0.1 + 0.3 -+ 0.1 + 0.2 -+ 0.1

1.4 + 0.3 1.6 + 0.1 1.1 +-0.1 0.7 + 0.1 0.5 +- 0.09 0.4 + 0.04 ND ND ND

Brain

1.7 1.9 1.8 1.4 1.2

_+ 0.02 _+ 0.02 _+ 0.1 _+ 0.09 _+ 0.1

1.4 + 0.4 1.6 + 0.1 1.2_+0.1 0.2 + 0.07 0.5 -+ 0.07 0.5 + 0.08 ND ND ND

Muscle

N D = Not detected *Multiply the values in the table by 10'* to get the actual tissue values: values for the blood arc expressed pcr ml rather than per gram. tResults arc for one animal less than is indicated for this time. ++This result is for ten animals. Values are means ± SEM for the nmnbers of animals given except where other~ise indicated. Radioactivity measurements for each tissue sample were made in duplicate.

No. of animals

Time after dosing

l'~C-activity (dpm × 10 "~/g)* in

Table 1. Distribution of ~4C-radioactivity following administration of ~'~C-labelled cheese to mice 1500 mg. 4.0 t~Ci) and to rats (2.2 g, 18 t*Ci)

gg

.a

v< ©

©

C L. GALLI et al.

316

Table 2. Excretion of 14C-radioactivity following administration of 1'*C-labelled cheeses to mice (0.5 g, 4.0HCi) and to rats 12.2 g, 18 HCi) Time after dosing (hr)

Percentage of administered radioactive dose recovered in Faeces + urine Mice + 1,8(3) + 2.1 (41 ± 2.2(6l + 7.5(111 _+ 3.3* (4) + 4.4"(41 ± 2.4* (41

2 4 8 16 32 64 96

10.6 13.8 19.2 55.7 63.7 61.1 59.0

4 8 16 32 64

Rats 2.1(31 4.0(3) 3.2(3) 3.6{3) 62.2 ± 5.2** (31 11.5 + 18.9 ± 21.0 + 67.0 +

Expired CO2

18.7 + 1.6(6) 19.4 ± 2.9(3i 24.3 + 5.1 (3)

6.7 +_ 0.413] 14.2 _+ 0.8(3i 26.6 + 3.6(3~ 27.9 ± 1.3(3) 30.3 4__ 5.3** (31

Values arc means ± SEM for thc number of animals indicated in brackets. Radioactivity measurements for each tissue sample were made in duplicate. Values marked with asterisks are not significantly different (P > 0.05. one-way analysis of wtriance)from the *16- or **32-hr value. In the rat, the liver was the only tissue where the radioactivity concentration/g tissue was equivalent to more than 0.1!~,, of the administered dose 6 4 h r after the ingestion of t'~C-labelled cheese. Recoveries of the 14C-label in the faeces, urine and expired air, expressed as a percentage of the dose. are shown in Table 2. Excretion of radioactivity by all routes from b o t h species was essentially complete within 32 hr (Table 2t. DISCUSSION In the present study we examined the distribution and the excretion in mice and rats of [l'~C]formaldehyde which was fed to the rats in grana cheese, made from milk with added [t4C]formaldehyde, a n d having the typical chemical and biochemical characteristics of cheese obtained by industrial dairies {Resmini et al. 1980b). The toxicokinetic profile appears to be almost identical in the two different species because of the similarity of the half-lives of the elimination phase, 27.8 hr a n d 26.4 hr. F u r t h e r m o r e the labelled cheese a n d / o r its metabolic products did not appear to concentrate preferentially in any of the tissues analysed in either species. After the administration of ~4C-labelled grana cheese to rats 270,, of the radioactivity was exhaled as ~'*CO2 during 16hr. This finding supports the evidence that in the cheese most of the formaldehyde reacts with milk proteins. It has previously been demonstrated that administration of free [x'*C]formaldehyde to rats, even ip, results in much faster evolution of >*CO2; in fact the peak ~'*CO2 expiration occurs after 1 hr (Neeley, 1964). F u r t h e r m o r e studies on an experimental grana cheese produced from milk with added [~'*C]fc, rmal-

dehyde demonstrated that almost all of the radioactivity was linked to casein which is the most a b u n d a n t milk protein (80!I,) and 1 2", bonded 1o the lipid fraction (Resmini el al. 1980b}. Analysis of the amino acids in the acid hydrolysate of a l~C-labelled grana cheese sample indicates that 90"o of the radioactivity is eluted in the basic fraction associated with the pool of basic amino acids and their methylated derivatives (Resmini. Saracchi, De Bernardi & Volonterio, 1980a). In addition chromatographic studies indicate that less than 5". of the radioactivity is in the form of [14C]methionine. However. after 10 months of ripening, x~hcn flcc H C t l O is no longer detectable. 50". of tfie radioactivity present in the cheese is linked to tire cascin and 50". is soluble at pH 4.6, probably b o u n d to catabolites of casein, to serum proteins or to minor c o m p o n e n t s such as small peptides tResmini el a/. 1980a}. Tire 14(,()~ rccovercd in the expired air of animals dosed with ~2('-Iabelled grana cheese probably originates flom Ihis sulublc fraction. Since no apparent toxic effecs haxc been observed limited methylation of food proteins after the reductive alkylation with formaldehyde may be useful as a protection method in processing and storagc ILee. Sen. Cliflbrd, W h i t a k e r & Feeney. 19781. It is now well established that the basic and acidic amino acid residues of certain proteins are methylatcd in cho (Vickery, 19721. Various mcth,,latcd amino acids are widely distributed in nature, and Ivsinc, histidine and arginine derivatives in particular tire ubiquitous in living organisms. Many of the methylated amino acid derivatives are also present in the free form in the urine and blood of animals and man (Paik & Kim. 1980a). Moreover. since methvlated amino acids cannot be reincorporated into protein in z,ico, some of these amino acids will find their wax into the kidney' for eventual disposal IPaik & Kim. 1980b). Although a study is in progress to identify the radioactive catabolites of ~4C-labelled grana cheese, we consider that no hazard would resuh from the ingestion of cheese containing the stable products of the reaction between formaldehyde and the amino acids of milk proteins or from their possible catabolites. After a single administration to rats and mice of a quantity of grana cheese equiwdent to the ingestion of 1.1 1.4kg of cheese by man. the concentration of radioactivity in the blood of either species, which was very likely in the form of methylated amino acids, was at all times several times below the concentration of a single methylated amino acid in the normal plasma of man or animals (Paik & Kim. 1980bL 4cknowh,dgements The authors thank Mb, s Daniela Pallamolla for secretarial assistance. This stud~ was supported by' a grant front "Consorzio per la tutela del li~rmaggio Grana Padano".

REFERENCES Auerbach C (1967). The chcmical production of mutations. Tile effect of chemical inutagens on cells and their genetic material is discussed. Science, N.Y. 158, l l4l. Auerbach C., Moutschen-Dahmen M. & Moutschen J. 11977). Genetic and cytogenitical effects of formaldehyde and related compounds. :",lutation Re,s. 39, 317. Chanet R. & yon Borstel R. C. 119791, Genetic effects of

Toxicity of grana cheese formaldehyde in yeast. III. Nuclear and cytoplasmic mutagenic effects. Mutation Res. 62, 239. Chaw Y. F. M.. Crane L. E.. Lange P. & Shapiro R. (1980). Isolation and identification of cross-links from formaldehyde-treated nucleic acids. Biochemistry, N.Y. 19, 5525. Collins C. J. & Guild W. R. (1968). Irreversible effects of formaldehyde on DNA. Biochim. biophys. Acta 157, 107. Della Porta G., Cabral J. R. & Parmiani G. (1970). Transplacental toxicity' and carcinogenesis studies in rats with hexamethylenetetramine. T , mori 56, 325. Della Porta G,, Colnaghi M. 1. & Parmiani G. (1968). Non-carcinogenicity of hexamethylenetetramine in mice and rats. Fd Cosmet. Toxicol, 6, 707. du Vigneaud V., Verly W. G. & Wilson J. E. (1950). Incorporation of the carbon of formaldehyde and formate into the methyl groups of choline. J. ,4m. chem. Soc. 72, 2819. Egle J. L., Jr (1972). Retention of inhaled formaldehyde. propionaldehyde, and acrolein in the dog, Archs em'ir. Hlth 25, 119. Epstein S. S., Arnold E.. Andrca J., Bass W. & Bishop Y. (1972). Detection of chemical mutagens by the dominant lethal assay in the mouse. Toxic. appl. Pharmac. 23, 288. Feldman M.Y. 11973). Reactions of nucleic acids and nucleoproteins with formaldehyde. Pro qr. Nucleic Acid Res. mol. Biol. 13, I. French D. & Edsall J. T. (19451. The reactions of formaldehyde with amino acids and proteins. Adv. Protein1 Chenl. 2, 277. Hurni H. & Ohder H. (1973}. Reproduction study with formaldehyde and hexamethylenetetramine in beagle dogs. Fd Cosmet. Toxieol. I I. 459. Johansson E. B. & Tj~.lve H. (1978). The distribution of [~aC]dimethylnitrosamine in mice. Autoradiographic studies in mice with inhibited and noninhibited dimethylnitrosamine metabolism and a comparison with the distribution of [14C]formaldehyde ' Toxic. appl. Pharmac. 45, 565. Kerns W. D. (1980). Long-term inhalation and carcinogenicity studies of formaldehyde in rats and mice. CIIT Conference on formaldehyde, Raleigh, NC, 20 21 Nov. Lee H. S.. Sen L. C., Clifford A. J.. Whitaker J. R. & Feeney R. E. (1978), Effect of reductive alkylation of the e-amino group of lysyl residues of casein on its nutritive value in rats. J. Nutr, 108, 687. Lewin S. (1966). Reaction of salmon sperm deoxyribonucleic acid with formaldehyde, Archs Biochem. Biophys. 113, 584. Malorny G., Rietbrock N. & Schneider M. (1965). The oxidation of formaldehyde to formic acid in the blood, a contribution to the metabolism of formaldehyde. Naunyn-Schmiedeber~t's Arch. exp. Path. Pharmak. 250, 419. Marsh G. M. (1982). Proportional mortality among chemical workers exposed to formaldehyde. In Proceedings of the Third Ammal C I I T Co~ferenee: Formaldehyde Toxic'it)'. Edited by J. E. Gibson. Hemisphere Publishing Corp. New York. In press. Martin C. N., McDermid A. C. & Garner R. C. (1978). Testing of known carcinogens and noncarcinogens for their ability to induce unscheduled DNA synthesis in HeLa cells. Cancer Res. 38. 2621.

I.¢ J

21 3

i

317

Neely W. B. (1964). The metabolic fate of formaldehyde~4C intraperitoneally administered to the rat. Biochem. Pharmac. 13, 1137. Obe G. & Beek B. (1979). Mutagenic activity of aldehydes. Drug Alcohol Depend. 4, 91. Paik W. K. & Kim S. (1980a). Natural occurrence of various methylated amino acid derivatives. In Protein Methj,lation. Edited by A. Meister, p. 8. John Wiley & Sons, New York. Paik W. K. & Kim S. (1980b). Free methylated amino acids. In Protein Methylation. Edited by A. Meister. p. 8. John Wiley & Sons. New York. Pruett J. J., Scheuenstuhl H., Michaeli D. & Nevo Z. (1980). The incorporation and localization of aldehydes (highly reactive cigarette smoke components) into cellular fractions of cultured human lung cells. Archs envir. Hlth 35, 15. Rcsmini P., Saracchi S., De Bernardi G. & Volonterio G. (1980a). lh L'aldeleide formica nel formaggio grana padano. L'industria de/ latte (3 4), 45. Resmini P., Saracchi S. & Motti G. (1980b). 1. L'aldeleide formica nel formaggio grana padano. L'industria del latte (3 4), 3. Rosenkranz H. S. & Leifer Z. (1980). Determining the DNA modifying activity of chemicals using DNA polymerase-deficient Escherichia coll. In Chemical Mutadens. Vol. 6. Edited by F. J. Senes & A. Hollander. p, 109. Plenum Press, New York. Ross W. E. & Shipley N. (1980). Relationship between DNA damage and survival in formaldehyde-treated mouse cells. Mutation Res. 79, 277. Swenberg J. A., Gross E. A.. Martin J. & Popp J. A. {1982). In Proceedings ol'the Third Annual C I I T Co~l~,rence: Formahtehyde Toxicity. Edited by J. E. Gibson. Hemisphere Publishing Corp., New York. In press. Swenberg J. A., Kerns W. D., Mitchell R. h, Gralla E. J. & Pavkov K. L. (1980). Induction of squamous cell carcinomas of the rat nasal cavity by inhalation exposure to formaldehyde vapor. Cancer Res. 40, 3398. Thomas J. O. (1976). Chemically-induced DNA protein cross-links. In Carcinogenesis and Radiation Biology. Edited by K. C. Smith. p. 193. Plenum Press, New York. Usdin V. R. & Arnold G. B. (1979). Transfer of Formaldehyde to Guinea-pig Skin. Gillette Research Inst. Report. Contract No. 78-391. Vickery H. B. (1972). The history of the discovery of the amino acids. II. A review of amino acids described since 1931 as components of native proteins. In Advances in Protein Chemistry. Vol. 26. Edited by C. B. Anfinsen. J. T. Edsall & F. M. Richard. p. 81. Academic Press. New York, Walrath J. & Fraumeni J. R. (1982). Proportionate mortality among New York embalmers. In Proceedings of the Third Annual C I I T Conference: Formaldehyde Toxicity. Edited by J. E. Gibson. Hemisphere Publishing Corp., New York. In press. Wong, O. (1982i. An epidemiologic mortality study of a cohort of chemical workers potentially exposed to formaldehyde with a discussion on SMR and PMR. In Proceedim,t s Oflthe Third Annual CITT Conference: Formaldehyde Toxicity. Edited by J. E. Gibson. Hemisphere Publishing Corp., New York. In press.