3 4 DISCUSSION

The results show that there was a correlation between the increase in MPO activity and lesion formation (damage score) in rat rectocolon treated with EtOH or EtOH+ TNBS. MPO activity thus seems to be a useful index of inflammation as previously reported [4681, and it has been found to be maintained for up to 3 weeks in EtOH+ TNBS-induced rectocolon damage in rats [4661. However, this trend was not shown in the concurrent treatment with DEM — a potent GSH depletor [4841 (Figure 3.2). A possible explanation for this observation may arise from the role of GSH as antioxidant that a minimum level of it is required to maintain normal polymorphonuclear leucocyte (PMNL) functions. This is because, as reported, auto-reactive oxidants derived from the extracellular release of the MPO/HgO;/halide system during PMNL activation are extensively involved in the auto-oxidative inhibition of PMNL functions, including

motility, and this inhibition of motility is reversible in the presence of antioxidancs such as ascorbate and cysteine [485, 486]. The maintenance of sulphydryl group (such as that of glutathione) in a reduced form at or above a critical minimal level may be important for motility [4851. In the present study, at 4 h, the marked depletion of mucosal GSH content induced by EtOH + DEM could render mucosal GSH level below the required minimum level for PMNL motility, thus leucocyte infiltration was inhibited. Consequently, MPO activity stayed at control level, even though high damage score was observed. The mucosal damage was derived from the initial lipid peroxidation and oxidative stress induced by EtOH + DEM. This initial damage might be prevented from perpetuating and propagating as a result of the initial inhibition of leucocyte infiltration, hence the subsequent absence of increased MPO activity even though the GSH content returned to normal or higher level (Figure 3.1). In this respect, depletion of GSH appears to give rise to beneficial effect. Furthermore, depletion of GSH has been shown to result in a marked increase in prostaglandin synthesis [48^, 4881. Cyclooxygenase, the enzyme which converts arachidonic acid to prostaglandin endoperoxides, is known to require the presence of hydroperoxide to initiate its activity; and depletion of GSH may enhance the availability of hydroperoxide, hence promote prostaglandin synthesis [4891. DEM has also been shown to induce a significant increase in 6-keto-PGF^^ (the degradation product of prostacyclin) and PGFg^ in gastric

mucosa [4371. Prostacyclin in turn has been shown to inhibit polymorphonuclear cell infiltration/in vitro [4901 and reduce leucocyte margination in the microcirculation [4911, hence DEM may indirectly modulate the accumulation of inflammatory cells. Alternately, a non-specific action of DEM on protein synthesis [4921 may account for the low MPO activity.

DEM conjugates with GSH and has a short duration of effect, since the mucosal GSH level increases rather rapidly after acute depletion [4931. This could account for the lack of depleting effect at 18 h and 7 day even though extra injections of DEM were given at day 3 and day 6 (Figure 3.1). This rapid recovery of GSH levels might

be due to the increased synthesis, and/or the increased blood supply to the tissue (could be due to the effect of prostaglandins), as erythrocytes and plasma are rich sources of GSH. GSH is known to be released from the liver into the circulation to maintain inter-organ GSH homeostasis [4941. GSH synthesis is thought to be regulated by end-product feedback inhibition [4951. Depletion of GSH would be expected to cause a compensatory increase in synthesis, hence the slight increase over control level in EtOH + DEM- and DEM-treated groups at 18 h after enema (Figure 3.1). The absence

of this effect in the EtOH-treated group could be due to the elevated MPO activity at 18 h, hence, the oxidation of GSH (Figure 3.1).

In the EtOH+TNBS-induced model, the lack of change in mucosal GSH content is perhaps not surprising in view of the prolonged time since the original insult. However, BSZ did produce a modest increase (not significant) in mucosal GSH content (Table 3.1). The trend of higher tissue GSH content in drug-treated groups than EtOH control in the in situ EtOH-perfused preparation (Figure 3.7.e) could be partly due to lesser GSH efflux (Figure 3.7.f) which in turn could be partly due to lesser blood loss (blood loss into the lumen, hence perfusate, as a result of mucosal damage) and lower oxidative stress in drug-treated groups. The former was as a consequence of protection of BSZ and SASP against cellular damage (Figure 3.3 & 3.7.c), while the latter, as a consequence of reactive oxygen metabolite scavenging effects of these compounds and a lower MPO activity (less inflamed) (Figure 3.5 & 3.7.b). SASP and its metabolite 5- aminosalicylic acid, and to lesser extent, sulphapyridine, have been shown to be free radical scavengers [71], but BSZ has yet to be been shown to be a free radical scavenger. As for the efflux of GSH in the in situ perfused preparation (Figure 3.6 & 3.7.f), the early efflux in drug-treated groups might be the consequence of transcellular transport of GSH to the lumen in response to the presence of xenobiotics. Alternately, it is possible that in the presence of BSZ or SASP, the conjugation of GSH with EtOH or oxidation of GSH induced by EtOH was somehow prevented to different degrees. The low perfusate GSH content during and immediately after EtOH challenge in the EtOH control group did not necessarily indicate reduced efflux of glutathione, as the assay method used did not measure glutathione in oxidized form (GSSG) or conjugated form that could be induced by EtOH. In later perfusion periods after EtOH challenge (in the absence of EtOH), the profile of efflux might reflect the blood loss to the lumen as a consequence of tissue damage (Figure 3.3). However, as discussed by Halliwell et a! (1992) [496], free radicals may be derived from drugs such as anti-inflammatory drugs, hence, the possibility that the radicals derived from both drugs (BSZ and SASP) might have led to the oxidation of GSH should not be ruled out. It should be pointed out that, in cell/tissue free and enzyme free conditions, ethanol and both drugs did not affect the measurement of GSH (results not shown).

Besides scavenging free radicals, GSH has, as a result of autoxidation, been shown to produce superoxide and hydroxyl radicals, hydrogen peroxide, and thinyl radical [404]. Depending on its concentration, GSH was found to enhance or inhibit hydroxyl radical formation [497]. Its role in inflamed rectocolon is complex, and its

effects may reflect the balance of its two contrary actions. In the present study, as discussed above, it seems that the reactive oxygen species generated by leucocyte activity may be less susceptible to GSH than that generated by acute EtOH insult (i.e. GSH is depleted more readily by acute ethanol insult than by leucocyte activity indicated by MPO activity). Furthermore, the tissue GSH pool can be restored rather quickly.

The addition of the protein-denaturing TNBS to ethanol has produced an inflammatory bowel model of greater duration than for ethanol alone. Treatment of an established inflammatory state (day 7) resembles more closely the clinical position. It is surprising that SASP, in this test, had no ameliorating activity but in fact slightly aggravated the inflammation, while BSZ reduced both rectocolonic lesions and MPO activity induced by EtOH 4-TNBS. The lack of effect of SASP on rectocolon damage in this model is in agreement with the work of Boughton-Smith et a! (1988) [465]. In contrast, both BSZ and SASP showed marked cytoprotective and anti-inflammatory effects in the acute inflammatory model represented by the in situ ethanol-perfused rat model. These findings are in agreement with the fact that both drugs are more successful in maintenance of remission [79, 80] rather than in treating severe active disease. BSZ has also been shown to have potent cytoprotective action on ethanol- induced gastric necrosis in rats [76].

CO

Ci

Ethanol Control Contro

M 2.0

Ethanol + DEM DEM Only

MPO