Nitrosamines hydroxylation

There is an industry trend to supply SBR certifiably free of volatile nitrosamines or nitrosatable compounds. This has generally been accomplished by replacing shortstop systems based on carbamates and hydroxyl amines with products that are not based on secondary amines or are secondary amines of high molecular weight, such as dibenzyldithiocarbamate. A more recendy issued patent for ESBR shortstop is based on isopropyUiydroxylamine, a primary amine that does not form nitrosamine (46). 

Biochemical Functions. Ascorbic acid has various biochemical functions, involving, for example, coUagen synthesis, immune function, dmg metabohsm, folate metaboHsm, cholesterol cataboHsm, iron metaboHsm, and carnitine biosynthesis. Clear-cut evidence for its biochemical role is available only with respect to coUagen biosynthesis (hydroxylation of prolin and lysine). In addition, ascorbic acid can act as a reducing agent and as an effective antioxidant. Ascorbic acid also interferes with nitrosamine formation by reacting direcdy with nitrites, and consequently may potentially reduce cancer risk. 

Alternative pathways of activation of nitrosamines, including 3-hydroxylation followed by sulfate conjugation and the formation of alkoxydiazen-ium ions are discussed. The formation of alkyldiazo-nium ions from trialkyltriazenes is presented to show that the formation of the putative ultimate carcinogens from nitrosamines can be studied in a system not requiring metabolic activation,... 

The above scheme satisfies much of the metabolic data however, some of it is speculative, and it is certainly incomplete. The evidence for the formation of the a-hydroxylated intermediate is circumstantial. The acetate ester of a-hydroxylated dimethylnitrosamine has been prepared (12.13) and has been found to be a potent, directly acting carcinogen (14). Other esters of a variety of a-hydroxylated nitros-amines have also been prepared (15). While it has been shown that DMN acetate is hydrolyzed to hydroxymethylmethyl-nitrosamine by an esterase enzyme, it has been pointed out that these derivatives of the a-hydroxylated nitrosamines also dissociate to N-nitrosoimmonium ions (15 16). 

All of these results support the o-hydroxylation hypothesis, but they do not prove it. No o-hydroxy nitrosamine or a derivative of one has ever been isolated as a metabolite of any nitrosamine. Nevertheless, a-hydroxylation is an attractive working hypothesis and it seems to account for... 

Although the o-hydroxylation hypothesis has been given considerable support, it is clear that nitrosamines with alkyl side chains longer than methyl present additional possibilities for biological activation. It was stated earlier that even the o-hydroxylation hypothesis is based on considerable circumstantial evidence. This means that the other possible pathways are even more speculative, and hence, much more research needs to be carried out before some of the ideas advanced below will gain acceptance, or be put to rest. 

It is known that nitrosamines with side-chains longer than methyl are metabolically hydroxylated on virtually every carbon. For example, the urinary metabolites in the rat of di-n-butylnitrosamine include the glucuronides of the following hydroxylated nitrosamines (42). 

In this particular instance, u) and m-l hydroxylation, which is then followed by further oxidation and chain degradation, are the principal reactions ( ) In the case of di-n-pro-pylnitrosamine both a- and 3-oxidation occur, with the latter being about 15% of the former (44) The a-hydroxy lation leads to the formation of the n-propyldiazonium ion (22), while the 3-hydroxylation results, at least in part, in oxidation to N-propyl-N-(2-oxopropyl)nitrosamine. Krdger and Bertrum (45) suggested that this product can be cleaved to methyl-... 

The purpose of the discussion of alternative pathways of nitrosamine activation (alternative to the a-hydroxylation hypothesis) is to demonstrate how fundamental organic chemistry may help classical metabolism studies to provide clues... 

The a-carbons of nitrosamines undergo enzymatic hydroxylation followed by oxidative cleavage leading to the formation of alkyldiazo hydroxides, alkyldiazonium ions and alkyl cations [34]. These cations are postulated to initiate the process of carcinogenesis in some cases by alkylating the base units of DNA [35, 36]. 

A method involving SPE was developed for the determination of ten A-nitroso amino acids in cured meat products. These compounds were derivatized with diazomethane followed by O-acylation of hydroxyl groups with acetic anhydride-pyridine reagent. The methyl esters and their acylated derivatives were separated by GC on a DB-5 fused silica capillary column and quantified with a TEA-CLD specific for the nitric oxide derived from the thermal denitrosation of nitrosamines recovery exceeded 75% at the 10 ppb level579. 

Hie mechanism by which the mixed function oxidase converts a nitrosamine into a carcinogenic compound is not clearly understood (]5). Hiese enzymic oxidations can occur at the alpha, beta, gamma, or omega position of an alkyl chain attached to the nitrosamine function. Regardless of whether or not these other oxidations are significant in carcinogenicity, it has been dorvon-strated that hydroxylation at the alpha position does indeed produce a carcinogen (33). Hie discussion which follows will be limited to those factors relative to oxidation at the a-position. 

This section will discuss experiments designed to synthesize phosphates of a -hydroxylated nitrosamines. These investigations shed some light on the thermal stability of a -substituted nitrosamines. 

It had been suggested a number of years ago that methyl alkyl nitrosamines are hydroxylated enzymatically on the methyl group to give a-hydroxymethylnitrosamines (2, 3). 

Thus, it is apparent that the initial a-hydroxylation of nitrosamine constitutes a possibly important pathway to produce the so-called proximate carcinogen. The a-hydroxylated nitrosamines have eluded direct isolation, although derivatives such as esters and ethers have been prepared by various groups (4, 5, 6, 7, 8). These materials, particularly g-acetoxydimethylnitrosamine (9), have been shown to be very potent carcinogens. 

The discovery of the anchimeric effect of the N-nitroso group led to several exciting possibilities. Kruger (12) postulated that if the side chain of nitrosamines is larger than ethyl, the compounds are degraded to methylating agents by B-hydroxyl-ation. He postulated that the function of this side chain... 

The ready alkylation of the bases derived from DNA suggests that it is likely that 6-hydroxylation followed by suitable conjugation may in fact, be an activation pathway for nitrosamines. 

Nitrosamines have the property of existing in two different stereochemical forms, if the groups on both sides of the nitrogen are different. In collaboration with Dr. Larry Keefer of the National Cancer Institute, we have utilized this property to determine which of the methyl groups in dimethylnitrosamine was hydroxylated. Using the specifically labeled Z-isomer (trideu-teromethyl group on the same side as the oxygen) we have determined that it is the methyl on the opposite side... 

Most of the over 400 N-nitroso compounds that have been tested for carcinogenic activity have been found to be carcinogenic [13, 39]. It is well known that the predominant initial metabolic activation pathway for N-nitrosamines is a-hydroxyl-... 

Substitution at the a-carbon with acidic group(s), fluoro group(s), or bulky group (s) has been shown to diminish or abolish their carcinogenicity. This is because the initial metabolic activation pathway for N-nitrosamines is a-hydroxylation. 

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