Vitamin A status appears to be an important factor in childhood mortality in areas where infectious diseases are responsible for substantial childhood morbidity and mortality, such as in developing countries. Supplementation with vitamin A reduces overall mortality and that attributable to diarrhea in young children in localities where vitamin A deficiency is a major public health problem (Ghana VAST Team 1993, Rahmathullah et al. 1990, West et al. 1991). The effects on childhood morbidity are less certain, although two studies (Barreto et al. 1994, Ghana VAST Team 1993) have found a decrease in the severity of diarrhea in children who are supplemented with vitamin A. 

The mechanism by which vitamin A supplementation reduces mortality is not clear. We previously determined that clinical vitamin A deficiency in rats reduced villus height and disaccharidase activity as compared to pair-fed controls, whereas subclinical vitamin A deficiency caused no changes in rat jejunal structure and function (Warden et al. 1996). Other investigators have found variable changes in intestinal structure and function in vitamin A deficient rodents (Ahmed et al. 1990, Chauhan and Kansal 1989, Majumdar and Ghosh 1987, Rojanapo et al. 1980, Singh and Krishnakantha 1987, Zile et al. 1977). Diarrhea was not observed in our vitamin A deficient rats (Warden et al. 1996), nor in other studies of intestinal structure and function in rodents with vitamin A deficiency alone (Ahmed et al. 1990, Chauhan and Kansal 1989, Majumdar and Ghosh 1987, Rojanapo et al. 1980, Singh and Krishnakantha 1987, Zile et al. 1977).

Vitamin A deficiency often occurs in environments in which gastrointestinal tracts are regularly insulted with pathogens and exogenous antigens. In light of both animal and epidemiological findings, we have hypothesized that vitamin A deficiency may exacerbate an insult to the gut, such as that caused by an infectious agent (Warden et al. 1996). In the only study to date to investigate this interaction, vitamin A-deficient mice were infected with rotavirus, resulting in a severe villus injury (Ahmed et al. 1990). This was not observed in uninfected, vitamin A-deficient mice, nor in vitamin A-sufficient, infected mice. Ahmed et al. (1990) concluded that the nonspecific mucosal barrier of the gastrointestinal tract was impaired in the presence of both vitamin A deficiency and infectious challenge. However, because that study used an infectious agent (i.e. rotavirus), it is difficult to determine whether the changes observed were caused by an impaired immune response secondary to vitamin A deficiency (Ross 1992) or to a local effect on the intestinal mucosa. Moreover, viral infections have been found to impair the vitamin A status of chickens (West et al. 1992). Thus we sought to use a noninfectious model of intestinal injury.

Methotrexate (MTX)⁶ is an antimetabolite widely used in cancer chemotherapy, which can cause intestinal mucosal injury (Loehry and Creamer 1969, Trier 1962). It inhibits the enzyme dihydrofolate reductase (DHFR) which is required for DNA synthesis and cell division (Jollivet et al. 1983). Alterations to mucosal surfaces, such as the gastrointestinal tract, and diarrhea are well-documented side effects of MTX in humans, attributable to MTX acting on the rapidly dividing mucosal cells (Jollivet et al. 1983, Loehry and Creamer 1969, Taminiau 1981, Trier 1962). MTX has been used to establish in rodents a reproducible model of small intestinal enteropathy characterized by diarrhea, villus atrophy and impaired digestive and absorptive function (Loehry and Creamer 1969, Pinkerton and Milla 1984, Takeuchi et al. 1989, Taminiau et al. 1980).

However, MTX injury is exacerbated by malnutrition and consumption of an elemental diet (Grossie et al. 1982, Harvey et al. 1984, Kehoe et al. 1986, Mihranian et al. 1984, Torosian et al. 1988), but alleviated by co-administration of vitamin A (Kosakai et al. 1991, Tsurui et al. 1990), suggesting that diet plays an important role in this model of injury. Thus the aim of this study was to examine the effect of vitamin A deficiency on the extent of jejunal injury caused by methotrexate.

MATERIALS AND METHODS

PF and +VA groups were also fed the same vitamin A deficient diet every second day. The PF group were pair-fed the amount consumed by the VA group on the previous two days, whereas the +VA group were allowed free access to the vitamin A deficient diet. Both PF and +VA groups were supplemented with vitamin A [Rovimix A 500W (70% retinyl acetate and 30% retinyl palmitate), gift of Roche Australia, Sydney, Australia] added to their drinking water every second day to deliver 37.5 µg/d (Study 1) or 75 µg/d (Study 2). The volume of water in which the vitamin A was provided was determined by that consumed over the previous two days. Additional water was provided if all water containing vitamin A was consumed.

All rats were housed in a single room in a conventional animal house, in wire bottom cages. A light:dark cycle of 12 h was maintained throughout the experiments, with the temperature kept at 20-22°C. Rats were monitored daily for signs of vitamin A deficiency (Warden et al. 1996) or diarrhea. These studies were approved by the Animal Care and Ethics Committee, The University of Newcastle.

Differences were considered significant at P < 0.05. Data were analyzed using Minitab Release 10 Xtra (Minitab, New York, NY). Rats were excluded from analyses if designated VA but with serum or liver retinol concentrations greater than three standard deviations from the group mean (n = 2), or died (n = 3); their pair-fed controls (n = 5); if incorrectly paired (n = 2); or if consistently unwell prior to MTX administration (+VA, n = 1).

RESULTS

Study 1: Supplementation with 37.5 µg vitamin A/d

Table 1. Serum and liver retinol concentrations of vitamin A deficient (VA) pair-fed (PF), and vitamin A sufficient (+VA) rats treated with vehicle or methotrexate (MTX)1 [View Table]

Study 2: Supplementation with 75 µg vitamin A/d

DISCUSSION

Vitamin A deficiency alone did not cause any changes in intestinal variables in this study, in concordance with our previous findings in subclinically vitamin A-deficient rats (Warden et al. 1996). However, treatment with MTX resulted in consistent, major injury to the VAMTX rats in all experiments. The same degree and consistency of injury was not observed in the vitamin A-sufficient groups that received MTX, which may be due to a greater susceptibility of the vitamin A-deficient rats to the toxic effects of MTX at this doseindeed, only vitamin A-deficient rats died after methotrexate treatment.

Malnourished rats exhibit signs of host toxicity at lower doses of MTX (Torosian et al. 1988) and have greater intestinal injury (Grossie et al. 1982, Mihranian et al. 1984, Torosian et al. 1988). It was essential to use pair-fed controls since we have previously found that consumption of the vitamin A-deficient diet results in reduced food consumption by vitamin A-deficient rats compared to vitamin A-sufficient rats from the first week of consuming such a diet (Warden et al. 1996). Similarly, subclinically deficient rats (40-42 days of dietary treatment) were studied to avoid the malnutrition associated with a longer duration of vitamin A deficiency (Warden et al. 1996). However, there were no differences in intestinal variables between vitamin A-sufficient, MTX-treated rats irrespective of whether they were pair-fed or had free access to the diet, suggesting that the degree of reduced food intake observed in our vitamin A deficient rats was not enough to exacerbate the MTX injury.

Pair-fed rats were also included to control for intake of any specific nutrient such as folate, whose metabolism is effected by MTX (Jollivet et al. 1983). The necessity for controlling for both macro- and micronutrients in experiments of this nature is exemplified by the study of Kehoe et al. (1986) who concluded that rats fed an elemental diet had greater injury following MTX administration than did those fed a pelleted diet. Although energy intake was controlled for, rats fed the elemental diet consumed substantially less vitamin A, which could have contributed to the findings.

The 100% difference in vitamin A supplementation did not result in significant differences for the intestinal variables measured when similarly treated groups were compared from these two studies. As expected, the higher level of supplementation resulted in greater liver retinol concentrations in all vitamin A-sufficient groups. However, the vitamin A-sufficient rats' liver retinol concentrations in Study 1 were less than that considered indicative of satisfactory vitamin A status (Batres and Olson 1987). Despite this, administration of MTX to these rats did not cause a more severe intestinal injury than in rats that received twice as much vitamin A. Although liver retinol concentration is considered a more satisfactory measure of vitamin A status than serum retinol (Underwood et al. 1979), liver retinol is mobilized more rapidly than extrahepatic retinol when insufficient vitamin A is available (Green and Green 1994), and models of retinol distribution in rats predict that relatively large pools of vitamin A may exist in extrahepatic and extravascular tissues when liver retinol is almost depleted (Green and Green 1994). An adequate store of intestinal mucosal retinol may explain why no differences were observed for intestinal variables in PF, PFMTX, +VA or +VAMTX rats irrespective of whether they received 37.5 or 75 µg vitamin A/d. Similarly it could also explain our previous findings of a significant difference in disaccharidase activity and intestinal morphology only in clinically vitamin A-deficient rats, despite both clinically and subclinically vitamin A-deficient rats having negligible serum and liver retinol concentrations (Warden et al. 1996).

The effect of vitamin A status on MTX-induced enteropathy appears to be due to an interaction between vitamin A and the action of MTX on the mucosa. However, it is unclear whether this is a direct interaction between these two agents, is mediated through other intestinal sequelae of vitamin A deficiency, or both. A reduction in crypt cell mitosis has been postulated to be the most likely cause for MTX-induced intestinal injury (Pinkerton and Milla 1984), although these authors suggest that there could also be a direct toxic effect of MTX on enterocyte protein and RNA synthesis. Whereas co-administration of vitamin A has been shown to prevent both the jejunal histological changes and the reduction in crypt cell de novo purine synthesis and pyrimidine salvage induced by MTX (Kosakai et al. 1991), we found no effect of vitamin A status on mucosal DNA concentration, whether or not MTX was administered. However, Zile et al. (1977), who also found no effect of mild vitamin A deficiency on mucosal jejunal DNA content, did find an increase of jejunal crypt cell cycle time associated with a decreased rate of DNA synthesis, and Chauhan and Kansal (1989) reported a reduction in small intestinal DNA content. Additionally, although small intestinal RNA concentration has been found to be reduced (Johnson et al. 1969) or unchanged (De Luca et al. 1969) in vitamin A deficiency, addition of retinoids stimulates small intestinal RNA synthesis in vitamin A-deficient rats (Johnson et al. 1969, Zile and Deluca 1970) and sucrase-isomaltase and alkaline phosphatase mRNA synthesis in small intestinal epithelial (IEC-6) cells (Nikawa et al. 1995).

Although the effect of vitamin A deficiency on DNA and RNA synthesis could provide an explanation for a direct interaction as a mechanism for the jejunal injury observed in the VAMTX rats, the agreement of our results with those of Ahmed et al. (1990), who infected vitamin A-deficient mice with rotavirus, suggests that other mechanisms should be considered. Indeed, vitamin A deficiency causes changes to mucosal barrier functions of the gastrointestinal tract which could predispose to or exacerbate an intestinal injury.

Vitamin A deficiency reduces mucosal immunity with decreased numbers of Peyer's patches and immunoglobulin-bearing cells in the gut-associated lymphoid tissue (Majumder et al. 1987), Peyer's patch T-cell proliferative response to mitogens (Majumder and Abdus Sattar 1987) and intestinal secretory IgA (Puengtomwatanakul and Sirisinha 1986). A reduction in goblet cell numbers in vitamin A deficiency (Ahmed et al. 1990, Rojanapo et al. 1980, Zile et al. 1977) and mucin production (Ahmed et al. 1990) has also been detected, suggesting an impairment of nonspecific mucosal defense. Macromolecule permeability is also increased in vitamin A deficiency (Gmoshinskii et al. 1987), indicating a defect in epithelial integrity. Furthermore, vitamin A deficiency results in decreased expression of Transforming Growth Factor (TGF) ₂ in lamina propria, surface epithelium and crypts of rat intestine, whereas administration of retinoic acid increases intestinal mucosal TGF₂ and TGF₃ expression (Glick et al. 1991). TGF have been implicated in the regulation of epithelial growth and differentiation (Gudas et al. 1994), and intestinal epithelial restitution (Dignass and Podolsky 1993), and indeed many of their actions on cells have been found to be similar to those of retinoids (Gudas et al. 1994).

The results of these studies may have not only clinical relevance for the prevention and minimization of diarrhea in children in developing countries in localities where both clinical and subclinical vitamin A deficiency are recognized as major public health problems, but also to areas where satisfactory vitamin A status is assumed, but is not necessarily present. In developed countries, diarrhea resulting from intestinal enteropathies either due to diseases such as human immunodeficiency virus (HIV) and inflammatory bowel disease, or as a result of treatments such as MTX and other chemotherapeutic agents that may injure the gastrointestinal tract, may be ameliorated by optimizing the patient's vitamin A status in addition to conventional treatment.

In summary, vitamin A-deficient rats have a more severe jejunal injury following methotrexate administration than do pair-fed controls. A 100% increase in dose of vitamin A caused a difference in liver retinol concentration in those rats receiving vitamin A, but in no intestinal variable studied, suggesting that locally available vitamin A may be important. These results have clinical implications in the prevention and management of diarrhea in both developing and developed countries.