Thiopurine antimetabolites, such as azathioprine (Aza) and 6-thioguanine (6-TG), are widely used in the treatment of cancer, inflammatory conditions and organ transplantation patients. Recent work has shown that cells treated with 6-TG and UVA generate ROS, with implied oxidatively generated modification of DNA. In a study of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in renal transplant patients, we provided the first in vivo evidence linking Aza and oxidatively damaged DNA. Using the hOGG1 comet assay we herein demonstrate high levels of 8-oxodG and alkali-labile sites (ALS) in cells treated with biologically relevant doses of 6-TG, or Aza, plus UVA. This damage was induced dose-dependently. Surprisingly, given the involvement of 6-TG incorporation into DNA in its therapeutic effect, significant amounts of 8-oxodG and ALS were induced in quiescent cells, although less than in proliferating cells. We speculate that some activity of hOGG1 towards unirradiated, 6-TG treated cells, implies possible recognition of 6-TG or derivatives thereof. This is the first report to conclusively demonstrate oxidatively damaged DNA in cells treated with thiopurines and UVA. These data indicate that Aza-derived oxidative stress will occur in the skin of patients on Aza, following even low level UVA exposure. This is a probable contributor to the increased risk of non-melanoma skin cancer in these patients. However, as oxidative stress is unlikely to be involved in the therapeutic effects of Aza, intercepting ROS production in the skin could be a viable route by which this side effect may be minimised.Azathioprine and UVA = 8-oxodG 3

We have developed new methods to minimize fluid shear during preparation of specimens for electron microscopy and to retain the mucous blanket that covers the tissue surface of the ileum in mice. We also used general stabilization by nonspecific antibodies to minimize the collapse of the mucous layer during dehydration for electron microscopy. These methods allowed us to visualize the gradual progression of the mucous blanket from a thin diaphanous layer in newborn animals to a very thick (ca. 50-,um), coherent structure in older animals that contained a mixed population of bacteria and protozoa. Some bacteria, notably filamentous forms, were patently anchored to the epithelial tissue but projected into the mucous blanket, whereas others clearly existed within the mucous blanket and were unattached to the epithelial surface. Similarly, some protozoa were firmly attached to the tissue surface, whereas others were suspended in the viscous mucous blanket. In an adult animal, the mucous blanket was a very thick layer which actually occluded most of the tissue surface and contained a rich variety of bacteria and protozoa.

Incorporation of purified phytohemagglutinin (PHA) lectins derived from red kidney beans (Phaseolus vulgaris) in the diet of weanling rats will cause growth failure, malabsorption of nutrients, and bacterial overgrowth in the small intestine. These effects are not caused by feeding a similar quantity of PHA to germfree rats. To define the morphological and bacterial changes on the mucosal surfaces of the jejunum, ileum, and cecum in greater detail, we pair fed two groups of weanling rats isocaloric, isonitrogenous diets with or without 0.5% PHA protein. On the jejunal surfaces of control rats, the mucous layer was a confluent covering with sparsely scattered bacteria and protozoa. In PHA-treated rats, the mucous layer was thin and discontinuous, and the microvilious surface of the tissue was extensively populated by bacterial cells of two distinct morphotypes-a gram-negative rod and a gram-positive coccobacillus. In all PHA-treated animals, these bacteria formed adherent monospecific or mixed adherent microcolonies on the tissue surface. Tissue damage was observed in PHA-exposed jejunal tissue as evidenced by vesiculation of the microvillous plasma membrane and by damage to the brush border membrane. On the ileal surfaces of control rats, there was a thick mucous layer within which small numbers of bacteria and protozoa were seen. Segmented filamentous bacteria were anchored in the tissue surface. In PHA-treated rats, the ileal surface was only incompletely covered by a mucous layer, and the overlying mucosal surface was extensively covered by large numbers of protozoan cells (predominantly Hexamita muris). Most of the ileal surfaces not covered by the mucous layer were occupied and virtually occluded by an overgrowth of these protozoan cells with occasional cells of Giardia muris and the tissue-associated segmented bacillus. In the ceca of control rats, the mucosa was incompletely covered by a discontinuous mucous layer and colonized by an unnamed Spirillum sp., other bacteria, and occasional protozoa. The cecal surfaces of PHA-treated rats retained most of their incomplete overlying mucous layer, which was heavily colonized by the same type of Spirillum sp. seen in untreated animals; intestinal crypts were colonized. These descriptive morphological studies demonstrate that exposure to purified PHA in the diet caused characteristic changes in the microbial ecology of the small intestine. The changes in microbial flora contributed to the malabsorption of nutrients in the small intestines of PHA-fed animals.

Bacterial translocation can be induced by the presence of an acute inflammatory focus in the peritoneal cavity. The translocation and inflammatory changes were associated with extensive loss of mesothelial cells. Nonetheless, these changes all resolved, indicating that the peritoneal cavity has a significant capacity to deal with such insults. A clearer understanding of the cellular and molecular events involved in the resolution phase could lead to improvements in the treatment of peritonitis.