Chemical exposure and intestinal function.

The particular substances that are ingested by individuals are the consequence of their environmental, residential, and occupational exposures. The possible effects of these exposures on intestinal functions can be examined by the evaluation of in vivo or in vitro exposure followed by an in vivo and/or in vitro monitoring of effects. Several examples of the in vivo exposure and in vitro monitoring approach are presented to demonstrate the consequences of oral exposure to either a heavy metal (arsenic), or a herbicide contaminant (2,3,7,8-tetrachlorodibenzo-p-dioxin) or a jet fuel propellant (hydrazine) and the subsequent measurement of either a particular metabolic pathway, or a cell-specific enzyme induction or the development of brush border enzymes are presented.

The particular substances that are ingested by individuals are the consequence of their environmental, residential, and occupational exposures. The possible effects of these exposures on intestinal functions can beexamined by the evaluation ofin vivo orin vitro exposure followed by an in vivo and/or in vitro monitoring of effects. Several examples of the in vivo exposure and in vitro monitoring approach are presented to demonstrate the consequences oforal exposure to either a heavy metal (arsenic), or a herbicide contaminant (2,3,7,8-tetrachlorodibenzo-p-dioxin) or a jet fuel propellant (hydrazine) and the subsequent measurement of either a particular metabolic pathway, or a cell-specific enzyme induction or the development of brush border enzymes are presented.
Human beings live in a very complex environment and are exposed to a multitude of natural and synthetic chemicals as illustrated in Figure 1. In addition to exposure by the skin and the respiratory system, the gastrointestinal tract provides a source ofcontact to a wide variety of chemicals which can be transported by a complex system (1). The kinds of substances include materials derived from ingested foods and liquids, food additives, food contaminants, drugs, particulate matter of inspired air which is swallowed, and bacterial and fungal species which proliferate within the intestines and may produce potent metabolic products. The substances ingested by an individual are the consequence of their environmental (water, air), residential (dirt, drugs) and occupational (dust, fumes) exposures.
The normal intestinal functions are summarized with examples in Table 1. An understanding of the basic principles of normal intestinal functions should permit greater appreciation for the unique roles of this organ in absorption and metabolism. In addition, an understanding of normal function may lead to better methods for detection of dysfunction and malabsorption. In the clinical setting, intestinal dysfunction (malabsorption syndrome) is monitored by gross inspection of the feces for fat, and measurement of serum carotene, calcium, albumen and cholesterol as well as monitoring the plasma prothrombin time. Intestinal malabsorption may be reflective of properties of a particular portion of the absorptive tract and of the etiological agent involved. Some of the parameters that have been developed for clinical diagnosis are just now being assimilated into animal studies in order to develop animal models for the evaluation of possibly harmful environmental exposures of the intestinal tract.
There is a rapidly growing body of knowledge concerning the effects of ingested substances on intestinal function. The approaches utilized in the examination of altered intestinal function include: in vivo exposure and in vivo (in situ) evaluation; in vivo exposure and in vitro evaluation; in vitro exposure and in vitro evaluation; in vivo exposure and a combination of both in vivo and in vitro evaluation. Examples of these approaches are presented in Table 2.
Several examples of in vivo exposure and in vitro monitoring will be examined in depth. These indepth examples of the in vivo exposure and in vitro monitoring approach are from the research performed in my laboratory. These examples are concerned with several aspects of intestinal function both in adult and developing animals and are summarized in Table 3.

Alteration of a Metabolic Pathway
An example of the in vivolin vitro approach which examines a metabolic pathway is provided by our work with prolonged oral arsenic exposure (9). In this study, the investigation focused on the utilization of pyruvate which is a key intermediate in intracellular metabolism. Our model system was the adult male Charles River CD rat given access to casein-based purified diet and to deionized drinking water containing 0, 40, or 85 ppm sodium arsenate (As 5+) for 3 weeks. This dosage regimen was chosen on the basis of a preliminary study (10) in which 85 ppm of sodium arsenate resulted in a significant (10%o) weight loss after 3 weeks ofexposure, (Fig. 2). No mortality occurred among any of the treatment groups during the course of the experiment.
The results of the respiration experiments indicated depressed state 3 (ADP present) respiration and decreased respiratory control ratios for pyruvate/malate-mediated respiration but not for succinate-mediated respiration. These effects were most pronounced in animals given the highest dose of 85 ppm arsenic (As5+). The ADP/O ratios were marginally depressed for both substrates at the highest arsenic dose level. These results indicated that mitochondrial oxidation of pyruvate/malate differed  from that of succinate. We then utilized a radiochemical assay technique (11,12) to investigate the mitochondrial enzyme pyruvate dehydrogenase, which exists both in an active (unphosphorylated) form and in an inactive (phosphorylated) form. Exposure of animals to arsenic for 3 weeks resulted in a decrease in both the basal (inactive) and total (active) pyruvate dehydrogenase activities as shown in Table   380 350 320 290-  4. This study suggested that in vivo exposure to arsenic decreases both the basal and total levels of pyruvate dehydrogenase activity in liver and intestine. In addition, the relative ease of activation appeared to increase after arsenic exposure. Because of the central role of pyruvate dehydrogenase in the mitochondrial utilization of pyruvate, the observed diminished pyruvate dehydrogenase activity after exposure to arsenic may/decrease the flow of acetyl-CoA into the tricarboxylic acid cycle as well as decrease the amount of fatty acid synthesis (Fig.  3). The selective effects of arsenic on mitochondria were also reflected in the decreased rate of oxidation of pyruvate by mitochondria isolated after treatment and in the abnormal ultrastructural architecture in these same animals (10).

Cell-Specific Enzyme Induction
A second example of the in vivo exposure and in vitro monitoring approach involved the study of the effects of an environmental agent, 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD), on cellspecific enzyme induction (13). TCDD is an extremely toxic contaminant produced during the commercial synthesis of the herbicide 2,4,5trichlorophenoxyacetic acid. A striking feature of the well documented toxicity of TCDD is the minute  quantity of TCDD required to produce pathogenic effects. Although the biochemical lesions underlying the observed toxicity of TCDD are not entirely understood, the activities of the UDP-glucuronyltransferase and aryl hydrocarbon hydroxylase are induced with nonlethal doses in rodent intestines (14)(15)(16). In our study, the TCDD-mediated induction of UDP-glucuronyltransferase was monitored in cells isolated from the tip and crypt regions of the intestines. The sensitivity of the isolated tip and crypt cells to this TCDD-mediated induction of UDP-glucuronyltransferase was determined by measuring time-dependent alterations in the level of enzyme induction and the concentrations of 14Cactivity in each of these cell types. The differential vibration method was used for the sequential removal of the intestinal cells (17). The effectiveness of this process was verified by morphological examination of the cells and by measurement of levels of appropriate marker enzymes (13). Adult female rats were fasted for 16 hr and then fed ad libitum after receiving a single oral dose at 8 ug/kg of 14C-TCDD. [The LD5o value for a single oral dose of TCDD is approximately 100 ug/kg (18).] p-Nitrophenol glucuronidation was measured 0, 3, and 10 hr and 1, 3, and 5 days after treatment. Basal activities of the crypt and tip cells were approximately 6.5 and 13 nmole p-nitrophenol conjugated/ min-mg protein, respectively (Fig. 4). UDPglucuronyltransferase activities were approximately the same as control values 3 hr following TCDD treatment. However, after 10 hr, tip cell activity increased by 20%X0 as compared to a 50o elevation of activity in crypt cells. The differential induction between the intestinal cell types became more pronounced by 1 day after TCDD administration, i.e., 200% increase in crypt cell activity and 50o increase in tip cell activity (Fig. 4). The greater sensitivity of the crypt cells to the TCDD-mediated effects of the intestine suggested a more general sensitivity of undifferentiated cells to inductive agents. Evidence for this greater sensitivity of the crypt cells to the actions of TCDD is provided by the fact that UDP-glucuronyltransferase induction in crypt cells occurs prior to the elevation of the tip cell enzyme activity (Fig. 4) and by the negative correlation between the level of UDPglucuronyltransferase induction and the measured levels of 14C-activity in these same cell preparations (Fig. 5). Estimates of the time required for the transit of the crypt cells to the tip of the villus, where they are sloughed off, are usually between 48 and 72 hr (19,20). During this period, the crypt cells lose their capacity to proliferate and become adapted to per- form absorptive functions. The levels of UDPglucuronyltransferase normally increase 2-fold in this transition period (Fig. 4). The time required for the appearance of marked UDP-glucuronyltransferase induction in the tip cells correlates well with the crypt to tip migration time of 2-3 days (Fig. 5).
Our observation that the levels of 14C-activity in both cell types were highest 3-5 days after treatment (Fig.  5) is consistent with the results of an earlier pharmacokinetic study (21). The elevated levels of 14C-activity observed in the isolated cells may, in part, be a reflection of the enterohepatic circulation of TCDD or a more polar metabolite. No attempts have been made at this time to determine the cellular distribution of the 14Cactivity in the isolated tip and crypt cells or the chemical nature ofthe 14C-labeled material. The role of enterohepatic circulation in the exposure of the FIGURE 6. Transmission electron photomicrograph of isolated intestinal cells prepared by vibration of rat duodenum. Method of preparation has been described in detail (13,17). 11,628x. intestine to metabolic products of ingested substances is receiving increasing attention. Of particular interest is the enterohepatic circulation of metabolites of hydrazine and its methylated derivatives.

Altered Intestinal Development
The last approach I will mention involves an investigation of altered intestinal function resulting from prenatal exposure to hydrazine and its methylated derivative, 1,2-dimethylhydrazine, which is a colon-specific carcinogen. The effects of prenatal exposure on normal intestinal enzyme development were examined (22) by monitoring three brush border enzymes, lactase, sucrase, and alkaline phosphatase, each of which has a unique metabolic function and, a different developmental pattern. After prenatal exposure to 1,2-dimethylhydrazine, the postnatal level of sucrase activity and all levels of alkaline phosphatase activity were elevated. In contrast, exposure to hydrazine diminished neonatal lactase, elevated postnatal and young adult sucrase, and elevated the neonatal and postnatal activities of alkaline phosphatase. Early changes resulting from an in utero exposure to these compounds may be an indication of an insult which is later expressed in pathological alteration to intestinal tissues such as tumor formation. Whereas altered enzyme activities are being utilized currently as indices of toxicity in general toxicology studies (9,13,23,24), the altera-tions we observed permit monitoring of altered enzyme activities in a specific organ after prenatal exposure to a carcinogen specific for that organ.
A model for studying prenatal exposure and intestinal function of offspring has been reported recently (3). In that instance fetal rats were exposed to a teratogen, either actinomycin-D, or hydroxyurea, or methotrexate, and then evaluated by the postnatal function tests of gastrointestinal transit time, rectal emptying, and gastric emptying (3).
Another study described the fetal and postnatal tissue distribution of several 14C-labeled chlorinated biphenyl after prenatal exposure (25). Chlorinated biphenyls that did not accumulate significantly in adipose tissue did accumulate dramatically in fetal intestine. Radioactivity was cleared rapidly from the intestine after parturition. When l-CB was administered at the 15th day of gestation, intestinal accumulation still did not occur until the 19th day of gestation, corresponding with the developmental onset of hepatic glucuronyltransferase. Approximately 10o of the fetal intestinal radioactivity following 1-CB treatment was in the parent compound, 5% was in 4-hydroxy-4-chlorobiphenyl, and 50o was in the glucuronide of 4-hydroxy-4-chlorobiphenyl. It is proposed that the chlorinated biphenyls are hydroxylated in the maternal compartment, and that the hydroxylated biphenyl metabolite is transferred across the placenta and conjugated by UDPglucuronyltransferase in the fetal liver. The glucuronide that reaches the intestine cannot be ex-  creted, but can be hydrolyzed by f3-glucuronidase.
The aglycone can then be absorbed into the fetal circulation and reconjugated in the fetal liver. We have monitored several intestinal enzyme development patterns in these offspring but found no conveniently altered indicator enzyme. This study suggests, however, a model for the study of fetal intestinal exposure as well as a mechanism for the metabolism of this class of chemicals by enzymes in the several compartments involved. At this point it may be appropriate to discuss the use of isolated cells for in vitro studies. We have observed that the tip cells (absorptive cells) prepared by the differential vibration technique maintain the characteristic columnar morphology of the absorptive intestinal cell (Fig. 6) of intact tissue. However, cell counts are difficult because of the aggregation of these cells either as a consequence of the mucus present or because the cells were isolated as clumps initially (Fig. 7). For studies that examine the in vitro uptake and metabolism of substances, we have developed another technique which involves a protease/EDTA treatment. The cells obtained by this enzyme/chelation method are monodispersed suspensions of absorptive cells (Fig. 8) and retain normal cellular membranous and subcellular organization as observed by transmission electron microscopy (Fig. 9). These cells have normal enzyme complements and are active metabolically.
Whereas these studies of the evaluation of in vivo exposure by in vitro monitoring may represent interesting and informative examples of the effects of environmental agents on intestinal functions, they provide, at best, only a beginning of future research in this area. There is a recent review which summarizes the information available on the gastroenterological responses to drugs and dietary test substances (1) (26). Together, these serve as a starting point for gastrointestinal toxicology.