Dehydroalanine and lysinoalanine in thermolyzed casein do not promote colon cancer in the rat.

Thermolysis of proteins produces xenobiotic amino-acids such as the potentially toxic lysinoalanine, and the alkylating agent, dehydroalanine, which have been considered possible health hazards. We observed that thermolyzed casein promoted aberrant crypt foci (ACF) and colon cancer growth in rats initiated with azoxymethane and speculated that promotion might be due to the formation of these compounds. To test this notion we first measured the concentration of the modified amino acids as a function of thermolysis time. The concentration of dehydroalanine in the casein paralleled the degree of promotion, that of lysinoalanine did not. We then tested diets containing foods with high levels of dehydroalanine (thermolyzed sodium-caseinate, cooked Swiss cheese) for their effect on ACF promotion. They decreased the number and/or size of ACF significantly, indicating that dehydroalanine did not promote, but protected rats against colon carcinogenesis. These results do not support the notion that lysinoalanine or dehydroalanine are a hazard with respect to colon carcinogenicity.

Toxicity of mixtures of nephrotoxicants with similar or dissimilar mode of action.

The toxicity of mixtures of chemicals with the same target organ was examined in rats using nephrotoxicants with similar or dissimilar modes of action. In a 4-wk feeding study, lysinoalanine, mercuric chloride, hexachloro-1,3-butadiene and d-limonene, each affecting renal proximal tubular cells but through different modes of action, were administered simultaneously at their individual lowest-observed-nephrotoxic-effect level (LONEL), no-observed-nephrotoxic-effect level (NONEL) and NONEL/4. Combined exposure at the LONEL resulted in increased growth depression and increased renal toxicity in male but not in female rats. Co-exposure at the NONEL produced only weak signs of toxicity (slightly retarded growth and increased renal weight), and rats co-exposed at the NONEL/4 did not show any treatment-related changes. The absence of an obviously increased hazard on combined exposure at the NONEL suggested absence of synergism and probably also of additivity. In a subsequent study the additivity assumption (dose addition) was tested, using the similarly acting nephrotoxicants tetrachloroethylene, trichloroethylene, hexachloro-1,3-butadiene and 1,1,2-trichloro-3,3,3-trifluoropropene. The compounds were given to female rats by daily oral gavage for 32 days either alone, at the LONEL and NONEL (= LONEL/4), or in combinations of four (at the NONEL and LONEL/2) or three (at the LONEL/3). Relative kidney weight was increased on exposure to the individual compounds at their LONEL and, to about the same extent, on combined exposure at the NONEL or the LONEL/3. As assessed by this endpoint, the renal toxicity of the mixtures corresponded to the effect expected on the basis of the additivity assumption. The other endpoints were not (or hardly) affected on combined exposure.

Subacute (4-wk) oral toxicity of a combination of four nephrotoxins in rats: comparison with the toxicity of the individual compounds.

In a 4-wk study, 10-wk-old Wistar rats were fed the nephrotoxins hexachloro-1,3-butadiene (HCBD), mercuric chloride, d-limonene and lysinoalanine either alone or in combination. These nephrotoxins damage epithelial cells of the proximal tubules, but by different mechanisms. Each chemical was given alone at a Minimum-Nephrotoxic-Effect Level (MNEL), and at a No-Nephrotoxic-Effect Level (NNEL). The combination was given at the MNEL, the NNEL and one-quarter of the NNEL of the individual chemicals. The individual nephrotoxins caused slight growth depression in males at the MNEL, but not at the NNEL, whereas the combination depressed growth slightly at the NNEL and severely at the MNEL. In females at the MNEL, only HCBD retarded growth; in contrast to the effect in males this was not aggravated by combined treatment. Nephrotoxicity was more severe in males fed the combination than in males given the nephrotoxins alone. The former showed decreased renal concentrating ability and moderate histopathological changes in the kidneys at the MNEL, and a dose-dependent increase in kidney weight and number of epithelial cells in the urine at the NNEL and the MNEL. The males treated with a single agent showed slightly increased kidney weights, and/or slight histopathological changes in the kidneys at the MNEL, and (with d-limonene only) epithelial cells in the urine at the NNEL and MNEL. In females, renal changes induced by the combination were not more severe than those observed with individual compounds. No adverse changes attributable to treatment were observed in rats fed the combination at one-quarter of the NNEL. In the present study, combined exposure to four nephrotoxins at their individual NNEL did not constitute an obviously increased hazard, indicating absence of synergistic interaction, whereas at the MNEL clearly enhanced (renal) toxicity occurred in males, although not in females.

Lysinoalanine: absence of mutagenic response in the Salmonella/mammalian-microsome mutagenicity assay.

Lysinoalanine (N epsilon-(DL-2-amino-2-carboxyethyl)-L-lysine) was tested for mutagenicity in the Ames Salmonella/mammalian-microsome mutagenicity assay. No mutagenic response was detected at doses up to 5 mg/plate when samples were pre-incubated without S-9 mix, nor when they were pre-incubated with S-9 mix prepared from Aroclor 1254-induced rat liver or kidney. The results indicate that the stereoisomers of lysinoalanine likely to be present in the greatest proportions in processed foods are not mutagenic in the Ames assay.

Protein-alkali reactions: chemistry, toxicology, and nutritional consequences.

Heat and alkali treatment of proteins catalyzes formation of crosslinked amino-acid side chains such as lysinoalanine, ornithino-alanine and lanthionine, and concurrent racemization of L-isomers of all amino acid residues to D-analogues. Factors that favor these transformations include high pH and temperature, long exposure, and certain inductive or steric properties of the various amino acid side chains. Factors that minimize crosslink formation include the presence of certain additives, such as cysteine or sulfite ions, and acylation of epsilon-NH2 groups of lysine side chains. Free and protein-bound lysinoalanine and D-serine induce nephrocytomegaly in rat kidney tissues. The presence of lysinoalanine and D-amino acid residues along a protein chain decreases its digestibility and nutritional quality. Understanding the factors that govern the formation of potentially harmful unnatural amino acid residues in food proteins and the toxic and nutritionally antagonistic action of these compounds in animals should lead to better and safer foods.

Food processing and storage as a determinant of protein and amino acid availability.

Protein is perhaps the most reactive of the major food components. During food processing, the essential amino acids, lysine, tryptophan, methionine and cyst(e)ine, may react with other food components causing a loss in amino acid bioavailability and sometimes a reduction in the digestibility of the whole protein molecule. This review first discusses some recent developments concerning protein-polyphenol reactions, racemization and lysinoalanine formation, and then describes the reactions of proteins and reducing sugars (the Maillard reaction) in greater detail. We report on the chemistry of the Maillard reaction, the nutritional and physiological properties of the newly-formed products and their metabolic transit in the rat. In practice, the Maillard reaction is by far the most important reaction of food proteins. It is especially important in milk products since these are the only naturally-occurring protein foods with a high content of reducing sugar. Lysine is the most sensitive amino acid to damage during processing and storage and its losses may be of nutritional significance to certain population groups, such as babies, who are often dependent on a single manufactured product as their sole source of nourishment.

Nonenzyme browning and its effect on protein nutrition.

The nonenzyme browning involves the thermal decomposition of sugars, the caramelization, the decomposition of oxi-acids, the so called "Maillard reaction" between amino acids and carbohydrates, the reaction between oxidized fats and proteins, and those alterations which take place by the alkaline treatment of proteins. The Maillard reaction is of secondary importance in the case of foodstuffs and fodders with low carbohydrate contents (meats, meat meal, fish meal). By the heat treatment, the sulphur-containing amino acids of proteins (cystine, methionine) are damaged primarily because of oxidation, but the decrease in the amount of threonine, serine, tryptophan, and lysine is observable too. According to the formation of enzyme resistant cross-links, the in vitro and in vivo digestibility of protein decreases after the heat threatment and the communication with oxidized fats. Besides the amino acids mentioned, the possibility of enzymatic break-off of leucine and isoleucine is reduced too. In the course of the heating of proteins the occurance of racemization has to be considered too (formation of alloisoleucine). The basic mechanism of the reaction between sugars and simple amino acids is already essentially explained: amino-acids break off after the formation glycosilamines and Amadori products but they are linked irreversibly to some, partly unsaturated decomposition products of sugars, types of 6 and 3 carbon atoms. The decrease in the biological usability of amino acids starts already with the Amadori products. The reactivity of the single amino acids depends on the number of carbon atoms, on the basicity, and on the polarity of the amino acid molecule. The especially highly reactive amino acids of proteins are (1) the essential lysine (because of its 6-HN2 group), (2) other types of basic amino acids, and (3) trypotphan (because of the lability of the indole ring), methionine, cystine and threonine. In the Maillard reaction of tryptophan the --NH--group of the indole ring is involved too. The Maillard reaction is highly influenced by the pH of foodstuffs or other agents. The reduction of pH which may be performed by the increase of fermentation in the baking industry, lessens the decomposition of lysine and tryptophan in proteins. The raise of pH in basic domain enhances the Maillard reaction up to a maximal value but a decrease may be observed when the pH is raised further on. In foodstuffs and in other solid protein-carbohydrate systems the increase of the moisture content generally enhances the Maillard reaction, the sensibility of the single amino acids to the changes int he moisture content is different. In the case of the alkaline treatment of proteins, we must reckon not only with the decomposition of single amino acids, first of all that of cystine by beta-elimination, but with the formation of some amino acid derivatives as lysinoalanine, lanthionine, and in ornithinoalanine too. Presently lysinoalanine is of toxicological importance as proved by experiences on rats...