It is axiomatic in bladder cancer research that urine is of fundamental importance (Clayson and Cooper 1970). Urinary tract carcinogens affect the urothelium via exposure through the urine rather than through the blood. Recently, however, urine itself has been identified as having relatively high concentrations of essential growth factors in urine, such as epidermal growth factor (EGF) (Momose et al. 1991). Animals exposed to carcinogens do not develop bladder tumors if the urothelium is not exposed to urine. Oyasu et al. (1981) showed that if a heterotopic bladder was exposed to a carcinogen, such as N-methyl-N-nitrosourea (MNU), followed by exposure to urine, bladder tumors developed. If the MNU was followed by exposure to saline, bladder tumors did not develop.

In addition to these direct influences of urine on bladder carcinogenesis, numerous indirect effects have been identified (Cohen 1995). These include pH, osmolality, volume, and others, many of which are highly responsive to variations in diet composition, diet intake and water consumption. It is these latter indirect influences of diet on the carcinogenic process that will be the focus of this article.

URINARY PHYSIOLOGY

The fact that there is a marked diurnal variation in most components of the urine poses difficulties technically in evaluating the role of these factors in carcinogenesis (Cohen 1995, Fisher et al. 1989). To avoid these difficulties, fresh voided urine is strongly recommended for studies on urinary effects in carcinogenesis.

The effect of these variations can be seen in Figure 1 (Fisher et al. 1989). As discussed in greater detail below, the effects of saccharin in the urine are dependent on urinary pH remaining above 6.5. When sodium saccharin is administered in the diet at high concentrations, urinary pH remains above 6.5 during the food ingestion period, but it rapidly decreases to below 6.5 when the rat stops eating when the lights come on. Calcium saccharin, in contrast, tends to keep the pH below 6.5 and has only marginal effects on the urothelium. If urinary pH is measured during the daylight hours, no differences are observed in urinary pH following feeding sodium saccharin vs. calcium saccharin.

As seen in Figure 1, the type of diet also has enormous effect on urinary pH. The AIN-76A diet produces a markedly acidic urine, with pH consistently below 6.0 even during periods of diet ingestion (Fisher et al. 1989). When fed in AIN-76A diet, neither sodium saccharin nor calcium saccharin produces a urine above the critical level of pH 6.5 at any time during the day, and no urothelial effects occur (Garland et al. 1989).

EFFECT OF DIET ON GENOTOXIC CARCINOGENS

EFFECT OF DIET ON NONGENOTOXIC URINARY BLADDER CARCINOGENS

Experiments performed in rodents generally make two basic assumptions with respect to extrapolation to humans (Cohen and Ellwein 1991): 1) the response in humans is reasonably similar to that in rodents and 2) the response at lower doses is similar to the response to the chemicals administered at higher doses in rodent experiments. For genotoxic chemicals, these two assumptions are reasonable, although the extrapolations may not be directly linear. For nongenotoxic substances, one or both of these assumptions may be incorrect. Urinary alterations greatly affect the potential of these chemicals to produce increased cell proliferation and therefore to effect carcinogenesis. Many of the experiments on the effects of diet on these chemicals have been performed utilizing various markers of increased cell proliferation as surrogate markers for tumor formation.

URINARY TRACT CALCULI

Calculi can form by a variety of processes in rodent urine (Clayson et al. 1995, Cohen 1995), including direct surgical implantation into the bladder lumen. Tumors eventually develop, with an increased incidence over time. Jull (1979) demonstrated that paraffin wax pellets implanted in mice produced an incidence of 10.6% after 12 mo, 26.8% after 18 mo and 53.8% by 24 mo.

Calculi can also be formed in the urinary tract by the administration of high doses of exogenous chemicals or by modification of normal physiologic processes in the body (Clayson et al. 1995, Cohen 1995). For example, surgically formed portacaval shunts lead to abnormalities in uric acid metabolism (Engelmann et al. 1987). Ultimately, urinary tract urate calculi form and tumors appear.

Substances that can lead to the formation of urinary calculi when fed in the diet are listed in Table 1 (Clayson et al. 1995). Some of these substances are essential ingredients in the diet (e.g., ascorbic acid, calcium), some are present in diet and are also formed during endogenous intermediary metabolism (e.g., glycine, uracil, uric acid), and some are foreign substances (e.g., melamine, fosetyl-al). The critical variable is the amount that ultimately is excreted in the urine and the concentration achieved. If an insufficient amount of chemical is ingested or formed endogenously to produce calculi, there is no toxicity, regeneration or carcinogenic effect. Carcinogenic effects present at high doses do not occur at low doses (Cohen and Ellwein 1991). This is obviously critical because many of the substances listed in Table 1 are essential for our survival.

Table 1. Chemicals administered at high doses that lead to urinary calculi in rodents [View Table]

It is also obvious from the list of substances in Table 1 that dietary factors and water ingestion can greatly influence their urinary concentration and thus the potential for precipitation. For example, increased intake of calcium simultaneously with substances that alkalinize urine can increase the risk of development of calcium oxalate or calcium phosphate calculi (Clayson et al. 1995, Cohen 1995). With respect to exogenous chemicals, modifying effects of the diet also can markedly affect the formation of calculi in the urine. For example, melamine administered at 3% of the diet in normal laboratory diet and water ingestion produce urinary calculi, proliferation and tumors (Ogasawara et al. 1995). However, if high doses of NaCl are administered along with the melamine, no calculi form and there is no tumorigenesis. Sodium chloride in the diet produces a marked increase in water ingestion and consequent increased urinary output of water. This results in a dilutional effect of the urine with decreased concentrations of the ingested exogeneous substances.

SODIUM SACCHARIN AND RELATED SALTS

Diet markedly affects the potential for saccharin and its various salts to cause an effect on the urothelium in rats (Ellwein and Cohen 1989). Administration of sodium saccharin in Agway Prolab 3200 diet produces a greater effect than administering it at comparable doses in Purina 5002 or NIH-07 diet (Fisher et al. 1989, Garland et al. 1989). When administered in AIN-76A diet, there is no effect whatsoever (Garland et al. 1989), because AIN-76A diet produces marked urinary acidification (see above). If the casein used in AIN-76A diet is replaced with ovalbumin as the protein source, the urinary pH is higher (unpublished observations).

The ultimate mechanism of carcinogenesis in rats secondary to sodium saccharin administration seems to be the formation of a calcium phosphate precipitate in the urine (Cohen et al. 1995). This is dependent on high concentrations of protein and apparently mucopolysaccharides (particularly heparan sulfate) that are present in rat urine. Concentrations of protein are greater in males than in females. Formation of this precipitate apparently requires a urinary pH of approximately 6.5 or higher. Thus, acidification of the urine, whether by administration of the AIN-76A diet or administration of sodium saccharin in other diets simultaneously with high concentrations of NH₄Cl, prevents the formation of the precipitate and prevents tumorigenesis of the bladder. The effects also appear to be strain related, with Sprague-Dawley rats being less susceptible than F344 rats. Sodium ascorbate administration in Oriental MF (Oriental Yeast, Tokyo, Japan) diet produces an effect in the rat urothelium similar to that of sodium saccharin, and the effect is greater in F344 rats than in Lewis rats (Mori et al. 1987). The opposite strain difference is observed when sodium ascorbate is administered in Clea CA-1 diet (Japan Clea, Osaka, Japan).

Feeding comparably high doses of a variety of other sodium salts (see Table 2) produces responses in the bladder of male rats that are qualitatively similar to those caused by sodium saccharin, although quantitatively there are variations (Cohen et al. 1995). Saccharin is the only non-natural substance on the list.

Table 2. Sodium salts that produce urothelial hyperplasia and increase bladder carcinogenesis when fed at high doses to rats [View Table]

Not only is the urothelial carcinogenicity of sodium saccharin and related sodium salts restricted to high doses (>1% of the diet), but the effect is specific to rats (Ellwein and Cohen 1989). Humans do not have the necessary urinary composition to generate the calcium phosphate-containing precipitate (unpublished observations), and thus they are unable to produce urothelial hyperplasia or carcinogenicity.

EFFECTS OF OTHER DIETARY COMPONENTS ON BLADDER CARCINOGENESIS

PROPOXUR, A URINARY MITOGEN

SUMMARY AND CONCLUSION