Epithelial Target Research Articles

Construction of an ecotropic retroviral vector expressing human insulin-like growth factor-I

There is increasing evidence that IGF-I plays an autocrine role in a wide range of human tumours, including, in particular, adenomas of the thyroid epithelium. To investigate this further, we set out to generate a retrovirus vector which would permit experimental manipulation of the expression of IGF-I in normal and neoplastic epithelial cells. We describe here the construction and validation of a high-titre ecotropic vector which transduces stable expression and secretion of human IGF-IA, as shown by analysis of mRNA and conditioned medium from rodent epithelial target cells. This vector should be a useful tool for assessing the contribution of abnormal IGF-I expression to the neoplastic phenotype.

Diversity of airway epithelial cell targets for in vivo recombinant adenovirus-mediated gene transfer.

A variety of pulmonary disorders, including cystic fibrosis, are potentially amenable to treatment in which a therapeutic gene is directly transferred to the bronchial epithelium. This is difficult to accomplish because the majority of airway epithelial cells replicate slowly and/or are terminally differentiated. Adenovirus vectors may circumvent this problem, since they do not require target cell proliferation to express exogenous genes. To evaluate the diversity of airway epithelial cell targets for in vivo adenovirus-directed gene transfer, a replication deficient recombinant adenovirus containing the Escherichia coli lacZ (beta-galactosidase [beta-gal]) gene (Ad.RSV beta gal) was used to infect lungs of cotton rats. In contrast to uninfected animals, intratracheal Ad.RSV beta gal administration resulted in beta-gal activity in lung lysate and cytochemical staining in all cell types forming the airway epithelium. The expression of the exogenous gene was dose-dependent, and the distribution of the beta-gal positive airway epithelial cells in Ad.RSV beta gal-infected animals was similar to the normal cell differential of the control animals. Thus, a replication deficient recombinant adenovirus can transfer an exogenous gene to all major categories of airway epithelial cells in vivo, suggesting that adenovirus vectors may be an efficient strategy for in vivo gene transfer in airway disorders such as cystic fibrosis.

Early effects of aldosterone on the basolateral potassium conductance of A6 cells.

Aldosterone increases the basolateral conductance in target epithelia. The basolateral membrane of tight epithelia contains two different types of K+ conductances (GK), a "resting" and a volume-activated GK. We have studied the early effects (at 4 hours) of 500 nmol/l aldosterone on the basolateral membrane GK of A6 cells (a Xenopus laevis kidney cell line), after the permeabilization of the apical membrane with amphotericin B. In the presence of a 97 to 3 mmol/l apical to basolateral K+ gradient, the "resting", inward rectifying GK was similar in control and aldosterone treated cells. In contrast, aldosterone induced a 2-fold increase of the volume-activated quinidine sensitive GK.

Adherence to respiratory epithelia by recombinant Escherichia coli expressing Klebsiella pneumoniae type 3 fimbrial gene products.

We examined the role of Klebsiella fimbrial types 1 and 3 in mediating adherence to human buccal and tracheal cells and to lung tissue sections. We found that clinical isolates of Klebsiella pneumoniae producing type 3 fimbriae and Escherichia coli HB101 containing a recombinant plasmid encoding expression of Klebsiella type 3 fimbriae (pFK10) demonstrated increased adherence to tracheal cells, trypsinized buccal cells, and lung tissue sections, in contrast to nonfimbriate and to type 1 fimbriate bacteria. Adherence by type 3 fimbriate bacteria was inhibited by purified type 3 fimbriae and Fab fragments derived from type 3 fimbrial-specific polyclonal immunoglobulin G. Type 3 fimbriae mediated attachment to the basolateral surface of tracheal cells and to the basal epithelial cells and the basement membrane regions of bronchial epithelia. Using an E. coli transformant (pDC17/pFK52), which expresses nonadherent P fimbrial filaments, along with the type 3 fimbrial adhesin (MrkD), we demonstrated that type 3 fimbrial attachment to respiratory cells was attributable to the MrkD adhesin subunit. Subsequent experiments demonstrated that the epithelial target of the type 3 fimbrial adhesin was most likely a peptide molecule rather than a carbohydrate. The results of this study demonstrate that, in vitro, the Klebsiella type 3 fimbrial adhesin mediates adherence to human respiratory tissue.

Aldosterone regulation of gene transcription leading to control of ion transport.

Aldosterone, like other steroid hormones, initiates its effects by binding to intracellular receptors; these receptors are then able to control the transcription of several genes. The products of these genes eventually modulate the activity of ionic transport systems located in the apical and the basolateral membrane of specialized epithelial cells, thereby modulating the excretion of Na+ and K+ ions. Considerable progress has been made recently in understanding these mechanisms and the structure of the proteins involved in these processes. A novel principle has been discovered to explain the selective effect of aldosterone on its target epithelia. These tissues exclude competing glucocorticoid hormones by the activity of the 11 beta-hydroxysteroid dehydrogenase to allow aldosterone, an enzyme-resistant steroid, to bind to its receptors. Aldosterone induces numerous changes in the activity of membrane ion transport systems and enzymes and cell morphology. Although the enhancement of Na,K-ATPase synthesis and the increase of the number of active Na+ channels in the apical membrane appear as both direct and primary effects, the mechanisms of the other effects remain to be determined. The knowledge of the primary structure of several elements of the aldosterone response system (e.g., mineralocorticoid receptor and Na,K-ATPase) allows us to understand abnormal regulation of Na+ balance at the molecular level and, potentially, to identify genetic alterations responsible for these defects.

Spontaneous cytotoxicity of human intraepithelial lymphocytes against epithelial cell tumors

Human intraepithelial lymphocytes are T cells primarily of the CD8+ phenotype located between intestinal epithelial cells. The cytotoxic and suppressor activities of these lymphocytes are largely unexplored. The spontaneous cytotoxic activity of these cells is evaluated in this study. Jejunal intraepithelial lymphocytes spontaneously lysed a variety of epithelial cell tumor lines (colonic and pancreatic adenocarcinomas and bladder epidermoid carcinoma) but not the highly natural killer-sensitive K-562 cells. Cold target inhibition studies showed that these lymphocytes preferentially bind the DLD-1 colonic adenocarcinoma cells rather than the K-562 cells. Pretreatment of the effector cells with interferon-γ did not change their cytotoxic activity. The cytotoxic cells are T lymphocytes (expressing CD2, CD3, and CD8). In contrast, the spontaneous cytotoxic activity of peripheral blood lymphocytes is directed against both epithelial cell targets and K-562 cells, is enhanced by interferon-γ, and is effected by natural killer cells (expressing CD2, CD16, and NKH1). Thus, the spontaneous cytotoxicity of intraepithelial lymphocytes differs from that of peripheral blood lymphocytes in their target cell restriction, lack of response to interferon gamma, and effector cell phenotype. Lymphocytes in the intestinal epithelium may have a novel and important role in recognizing and destroying transformed epithelial cells and colon cancers.

Deletion of the growth factor gene related to EGF and TGF alpha reduces virulence of malignant rabbit fibroma virus.

The role of the epidermal growth factor homologue in malignant rabbit fibroma virus (MRV) pathogenicity was investigated by constructing a viral growth factor deletion mutant (MRV-GF-). Since MRV is a recombinant virus with a myxoma virus background but possesses some terminal sequences derived from Shope fibroma virus, the growth factor gene in MRV is in fact identical to Shope fibroma growth factor (SFGF). Although no significant differences were detected in the in vitro characteristics of MRV and MRV-GF-, a pronounced attenuation was observed after inoculation of the test rabbits with MRV-GF-. Animals infected with wild-type MRV uniformly developed a fatal syndrome involving disseminated tumors accompanied by purulent conjunctivitis and rhinitis. In contrast, although MRV-GF- recipients developed similar initial signs of the MRV disease syndrome, 75% of these animals completely recovered from the viral and secondary bacterial infections and became immune to subsequent MRV challenge. Tumors in MRV-GF- recipients displayed earlier and more prominent inflammatory reactions than their wild-type MRV counterparts and contained fewer proliferating cells. Squamous metaplasia and hyperplasia of target epithelia were less pronounced in MRV-GF- than in MRV infection. We conclude that SFGF is a major virulence factor in MRV infection and is responsible for at least some of the cellular proliferation observed at tumor sites. In addition, the diminished ability of MRV-GF- to cause hyperplasia in nasal and conjunctival epithelia may decrease the extent of gram negative bacterial overgrowth as compared to the parental virus and hence contribute to the dramatic reduction in the lethality of MRV-GF- infection.

Neutrophil-mediated damage to human gingival epithelial cells.

Polymorphonuclear leukocytes (PMNs) have been implicated in the pathogenesis of inflammatory gingivitis and periodontitis. To further study the role of PMNs in mediating gingival injury, we cocultured these cells in vitro with monolayers of human gingival epithelial cells. Scanning electron microscopy revealed that the epithelial cells were homogeneous and SDS-PAGE/immunoblot analysis identified the presence of keratins K3, K13 and the K6/16 pair which authenticated the oral origin of the cells. Injury to the gingival cells was determined by scanning electron microscopy and measurement of cell detachment and cytolysis. Unstimulated PMNs produced minimal lysis or detachment, but PMNs stimulated by phorbol myristate acetate produced marked epithelial cell detachment without lysis, which was time- and PMN-dose-dependent. Supernatants of activated PMNs were similarly effective, indicating that the mediator was a stable soluble substance. Elastase and cathepsin G, two neutral proteases of PMN origin, produced time- and concentration-dependent detachment of gingival epithelial cells, suggesting that these enzymes may mediate this form of injury. In other studies, gingival epithelial cells were exposed to PMN myeloperoxidase (MPO), chloride and glucose plus glucose oxidase (GO) as a hydrogen peroxide (H2O2) generating system. The toxic oxygen species produced by this system caused lysis of the epithelial targets which was dependent on the duration of incubation and the concentrations of MPO and GO. Azide, an inhibitor of MPO, and catalase, a scavenger of H2O2, inhibited the lytic activity of this system. Scanning electron micrographs of gingival epithelial cells cocultured with activated PMNs showed lifting of the cells from the plating surface, while target cells attacked by the MPO system revealed extensive damage of cell membranes. These studies indicate that activated PMNs cause nonlytic detachment injury to gingival epithelial cells which may be mediated by digestion of their extracellular matrix by granule neutral proteases. Furthermore, PMN MPO is capable of generating toxic oxygen species which can lyse these epithelial cells. Collectively, these actions could have profound adverse effects on the function and integrity of the gingival epithelium.

CYTOPATHOGENICITY OF ENTAMOEBA HISTOLYTICA TO HUMAN INTESTINAL EPITHELIAL CELLS: INHIBITION BY MONOCLONAL ANTIBODIES AND SERA FROM AMOEBIC PATIENTS

Interactions of pathogenic Entamoeba histolytica (HM 1) with human intestinal epithelial cells (Henle-407) were investigated. The E. histolytica trophozoites adhered and cytolysed 87% of cultured epithelial cell monolayers. A significant (P less than 0.001) inhibition of cytopathic effect of amoebic trophozoites pretreated with monoclonal antibodies to a 29 kDa surface associated protein suggested utilization of the 29 kDa surface protein in recognition and cytolysis of epithelial target cells. The polyclonal sera from treated patients of amoebic liver abscess and anti-amoebic hyperimmune serum inhibited cytopathogenicity to a greater degree (P less than 0.001) than did the monoclonal antibodies. The data thus suggest involvement of several amoebic molecules in exercising cytopathogenicity to epithelial cells.

Multiple functions for NGF receptor in developing, aging and injured rat teeth are suggested by epithelial, mesenchymal and neural immunoreactivity

We have used immunocytochemistry to analyse expression of nerve growth factor receptor (NGFR) in developing, aging and injured molar teeth of rats. The patterns of NGFR immunoreactivity (IR) in developing epithelia and mesenchyme matched the location of NGFR mRNA assayed by in situ hybridization with a complementary S35-labeled RNA probe. The following categories of NGFR expression were found. (1) There was NGFR-IR in the dental lamina epithelium and in adjacent mesenchyme during early stages of third molar formation. (2) NGFR-IR nerve fibers were posterior and close to the bud epithelium. (3) During crown morphogenesis NGFR expression was prominent in internal enamel epithelium and preodontoblasts; it faded as preameloblasts elongated and as odontoblasts began to make predentin matrix; and it was weak or absent from outer enamel epithelium, the cervical loop, and differentiated ameloblasts and odontoblasts. (4) When NGFR-IR nerve fibers entered the molars late in the bell stage, they innervated the most mature peripheral pulp and dentin in an asymmetric pattern which correlated more with asymmetric enamel synthesis than with mesenchymal NGFR-IR distribution. (5) The mesenchymal pulp cells continued to have intense NGFR expression in adult teeth, especially near coronal tubular dentin. (6) The pulpal NGFR-IR decreased in very old rats or subjacent to reparative dentin (naturally occurring or experimentally induced). (7) During root formation, the preodontoblasts had NGFR-IR but most root mesenchymal cells and Hertwig's epithelial root sheath did not. This work suggests that there are important epithelial and mesenchymal targets of NGF regulation during molar morphogenesis that differ for crown and root development and that do not correlate with neural development. The continuing expression of NGFR-IR by pulpal mesenchymal cells in adult rats was most intense near coronal odontoblasts making tubular dentin; and it was lost during aging, or subjacent to sites of dentin injury that caused a phenotypic change in the odontoblast layer.

Characterisation of the effector cells responsible for the in vitro cytotoxicity of blood leucocytes from aphthous ulcer patients for oral epithelial cells.

This study was designed to identify the cells responsible for the spontaneous cell mediated cytotoxic effect (SCMC) exerted by peripheral blood leucocytes from patients with recurrent aphthous ulceration, towards cultured oral epithelial cells. Peripheral blood leucocytes from recurrent aphthous ulceration patients exerted a significantly greater (p less than 0.01) degree of cytotoxicity towards the oral epithelial target cells than did peripheral blood leucocytes from healthy control subjects, or from patients with non-specific ulceration. Depletion of CD-5 positive cells (T-lymphocytes) resulted in a significant decrease in the SCMC in aphthous patients. Depletion of CD-16 positive cells (NK-cells) produced no significant change in cytotoxicity. T-lymphocytes, therefore, appear to be intimately involved in the in vitro SCMC effect in recurrent aphthous ulceration.

Direct Cytotoxicity of Polymorphonuclear Leukocyte Granule Proteins to Human Lung-derived Cells and Endothelial Cells

Neutrophils, in the course of defending the host against microbial invasion, release a potent arsenal of proteins that can potentially damage host tissues. Defensins are major peptides of human polymorphonuclear leukocyte (PMN) granules and are both broadly microbicidal and cytotoxic to several tumor cell lines. To determine whether these peptides could play a role in neutrophil-mediated lung injury, we examined the cytotoxicity of defensins and other PMN granule proteins in a chromium release assay with human lung-derived cell lines MRC-5 (lung fetal fibroblast), A549 (lung adenocarcinoma with features of alveolar epithelium), and primary cultures of human umbilical vein endothelial cells (HUVEC). Crude fractionation of an acid extract of human PMN granules yielded four fractions A-D. Only fraction D (containing mostly defensins) was significantly cytotoxic to all three target cells. In contrast, fraction A (containing myeloperoxidase and lactoferrin) and fraction C (containing lysozyme) had little effect, and fraction B (containing chiefly cathepsin G and elastase) was only injurious to endothelial cells. The cytotoxicity of whole PMN granule extracts on pulmonary epithelial and fibroblast targets could be completely accounted for by their defensin content. Fraction D- and defensin-mediated cytotoxicity was concentration dependent, required at least 10 to 12 h to become manifest, and was inhibited by serum. The role of these peptides in lung damage during acute and chronic inflammation deserves further study.

Aldosterone increases the apical Na+ permeability of toad bladder by two different mechanisms.

The aldosterone-induced augmentation of Na+ transport in toad bladder was analyzed by comparing the hormonal actions on the transepithelial short-circuit current and on the amiloride-sensitive 22Na+ uptake in isolated membrane vesicles. Incubating bladders with 0.5 microM aldosterone for 3 hr evoked more than a 2-fold increase of the short-circuit current (because of the activation or insertion of apical amiloride-blockable channels) but had no effect on the amiloride-sensitive Na+ transport in apical vesicles derived from the treated tissue. A longer incubation (e.g., 6 hr) produced an additional augmentation of the short-circuit current, which was accompanied by about a 3-fold increase of the channel activity in isolated membranes. The stimulatory effect of aldosterone sustained in vesicles was inhibited by the antagonist spironolactone (present at 1000-fold excess) and the protein synthesis inhibitor cycloheximide (1 microM). In addition, triiodothyronine and butyrate, previously reported to partly inhibit the aldosterone-induced increase in short-circuit current, blocked the hormonal effect in vesicles. It is suggested that aldosterone elevates the apical Na+ permeability of target epithelia by two different mechanisms: a relatively fast effect (less than or equal to 3 hr), which is insensitive to triiodothyronine or butyrate and is not sustained by the isolated membrane, and a slower or later (greater than 3 hr) response blocked by these reagents, which is preserved by the isolated membrane. The data also indicate that these processes are mediated by different nuclear receptors.

Lysis of colonic epithelial cells by allogeneic mononuclear and lymphokine activated killer cells derived from peripheral blood and intestinal mucosa: evidence against a pathogenic role in inflammatory bowel disease.

A sensitive 4 h 51Cr-release cytotoxicity assay has been developed using as targets colonic epithelial cells obtained by Dispase-collagenase digestion of resected mucosa or colonoscopic biopsies. Peripheral blood mononuclear cells (MNC) from most healthy donors showed low, but significant levels of cytotoxicity for normal epithelial cell target cells of 8.7 (4.4) % (mean (SD] and similar levels were found in 14 ulcerative colitis (6.5 (4.4) %) and 16 Crohn's disease (6.2 (5.2) %) patients. Neither drug therapy nor disease activity influenced the results. The sensitivity of colonic epithelial cells isolated from inflamed and histologically normal mucosa to lysis by peripheral blood MNC from a single donor was not affected by the underlying disease. Anti-epithelial cell activity did not correlate with anti-K562 activity and the cytotoxic cell was plastic non-adherent and Leu-11b-. None of 15 MNC populations isolated from mucosa of normal, tumour bearing, or chronically inflamed intestine exhibited significant lysis of colonic epithelial cells despite killing of K562 target cells in 10. Lymphokine activated killer (LAK) cells, generated by interleukin-2 stimulation in vitro of nine intestinal and seven peripheral blood MNC populations, exhibited high levels of lysis of K562 cells but, on every occasion, failed to lyse colonic epithelial cells. These data indicate that spontaneously cytotoxic or LAK cells are unlikely to play a role in the generation of colonic epithelial cell injury by direct cytotoxicity in inflammatory bowel disease.

HSV-1-infected Oral Epithelial Cells are Targets for Natural Killer Cells

A mechanism of cell-mediated immunity was investigated to determine whether natural killer (NK) cells were able to lyse antigenically-altered epithelial cell targets. Using standard four-hour chromium-release assays, we tested human peripheral blood lymphocytes against autologous untreated epithelial cells and autologous HSV-1-infected epithelial cells and calculated the percentage of lysis. With adherent cell-depleted peripheral blood, only epithelial cells infected with virus were lysed (p = 0.009). Evidence that NK cells were responsible for the lysis exists because: (1) peripheral blood lymphocytes were able to lyse allogeneic as well as autologous virus-infected cells; (2) when NK cells were depleted with the lysosomotropic drug L-leucine methyl ester, cytotoxicity against infected targets was abrogated; and (3) depletion of NK cells by the monoclonal antibody Leu-11b, plus complement, also eliminated cytotoxicity against virus-infected targets. Additional evidence suggests that lysis of targets does not involve antibody-dependent cellular cytotoxicity. These findings indicate that NK cells have the potential to perform a similar in vivo immunologic role in the oral cavity and initiate cell-mediated lysis of virus-infected epithelial cells.

Models of aldosterone action on sodium transport: emerging concepts.

This article will detail models of aldosterone action in target epithelia such as kidney and toad bladder with respect to Na transport. The renal target cell responsible for aldosterone-dependent Na reabsorption is considered to be the cortical collecting tubule (CCT), while that responsible for acid secretion is characterized by the inner strip of the outer medullary collecting duct.1 An analogous situation is found in toad bladder, with one cell type (granular cell) responsible for aldosterone-dependent Na reabsorption while another (mitochondrial-rich cell) modulating acid secretion in response to steroid.2 With respect to K transport, these two tissues differ, in that aldosterone does not modify the secretory rate of K in the toad bladder due to a vanishingly small apical membrane K permeability, while in the mammalian kidney, aldosterone both increases the urinary excretion of K and the secretion of K by CCTs under steady-state conditions.3–5 With regard to the Na reabsorbing cell, the following paragraphs will both detail existing data and the various working hypotheses currently being evaluated to interpret that data with respect to the means by which aldosterone alters (a) luminal membrane Na permeability, and (b) NaK ATPase activity. In addition, the possible roles of energy and phospholipid metabolism in this process will be discussed. Since some actions of aldosterone parallel those of ADH (antidiuretic hormone), data on both hormones will be contrasted as appropriate in order to help narrow the possible explanations into a cohesive model for the Na reabsorbing cell.

Evidence for direct action of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) on thymic epithelium

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) acts on selected targets within the immune system to produce a characteristic profile of pathologic responses typified by thymic atrophy, suppressed cellular immunity, and inhibition of antibody production to T-lymphocyte-dependent antigens. Studies in inbred mice differing in sensitivity to TCDD indicate that TCDD-induced thymic atrophy is mediated by a receptor protein (designated the Ah receptor). To study the cellular and molecular basis for TCDD-induced thymic atrophy, primary cultures of thymic epithelial (TE) cells were established from C57BL 6 mice, a strain sensitive to TCDD. Treatment of TE monolayers with TCDD (0.1 to 10 n m) resulted in the altered maturation of cocultured syngeneic thymocytes as judged by suppression (40% of control at 10 n m TCDD) of TE-dependent responsiveness of thymocytes to the mitogens concanavalin A and phytohemagglutinin. TE-conditioned medium enhanced the mitogen responsiveness of thymocytes three- to four-fold; however, the enhanced mitogen response mediated by the TE-conditioned medium was not suppressed in thymocytes incubated in medium collected from TCDD-treated cultures or in TE-conditioned medium to which TCDD (10 n m) had been added directly. The suppression of TE-dependent maturation of thymocytes was concentration dependent (EC 50 ∼ 1 n m) and stereospecific, suggesting involvement of the Ah receptor. The Ah receptor in cytosol fractions from cultured TE cells was measured directly and was found to be present at a concentration 3 and 3.5 times greater than that measured in whole thymus and thymocytes, respectively. The results of this study indicate that TCDD can act directly on epithelial target cells in the thymus: one consequence of this action appears to be the altered thymus-dependent maturation of T-lymphocyte precursors, mediated through direct cell-cell contact between thymocytes and TE cells.

Cellular pH and the ADH-induced hydrosmotic response in different ADH target epithelia

The hydrosmotic response elicited by oxytocin in the frog skin epithelium (Rana esculenta) was reversibly inhibited by 70% when the medium pH was reduced to 6.2 by CO2 bubbling on the serosal side. On the contrary, the response to 8-bromo cyclic AMP (8 Br-CAMP) was not affected by medium acidification, even after corion removal. In other experiments intracellular pH was measured, employing the dimetyl-oxazolidine-dione distribution technique, in frog urinary bladder and the isolated frog skin epithelium. As previously observed in the case of oxytocin, 8 Br-CAMP increased intracellular pH in frog urinary bladder. Incubation with oxytocin also augmented the intracellular pH in the isolated frog skin epithelium but 8 Br-CAMP did not modify cell proton concentration in this tissue. From previous and present results it can be summarized that: 1) The intracellular alkalinization effect elicited by oxytocin addition and the inhibition in the hydrosmotic response induced by medium acidification were qualitatively similar in both tested target epithelia. 2) On the contrary, a post cyclic AMP step sensitive to changes in intracellular pH was not observed in frog skin, as is the case in frog urinary bladder. 3) The 8 Br-CAMP induced intracellular alkalinization effect was only observed in frog urinary bladder.

Chymotrypsin, ileal chloride transport, and neurotransmitters.

Electrical field stimulation (EFS) depolarizes nerves and causes chloride secretion by mucosa of rabbit ileum mounted in a flux chamber. To test the hypothesis that the transmitter is a peptide, we determined whether the EFS response was prevented by the endopeptidase chymotrypsin (CT). Serosal, but not mucosal, addition of CT (200 micrograms/ml) reduced the short-circuit current (Isc) response to EFS by 90% or more. CT also reduced Cl absorption by decreasing the mucosal-to-serosal flux, but it did not affect net Na absorption. CT prevented the response to vasoactive intestinal polypeptides, but the response returned when CT activity was eliminated. The response to EFS did not return, however, implying that CT damaged cells that released transmitter or epithelial target cells. CT reduced the Isc response to serotonin by 69% and to A23187 by 10% and did not affect the theophylline response. We conclude that 1) the effects of CT on cell function limit its usefulness in identifying peptide neurotransmitters in epithelium, 2) CT irreversibly inhibits ion transport responses to EFS and to serotonin, and 3) CT reduces absorption of Cl probably by affecting a calcium pathway that modifies Cl transport.

Evaluation of Mixed Exposure to Carcinogens and Correlations of in vivo and in vitro Systems

The biological evaluation of air pollutants is an example of the difficulties of evaluating the effects of mixed concurrent exposures to multiple agents, such as combinations of carcinogens with other carcinogens of the same or different chemical class, with incomplete carcinogens and cocarcinogens, with particulate materials and other factors that modify tissue distribution and retention, and with modifiers of metabolic pathways of activation and detoxication. A research approach is outlined to investigate such interactions in a series of biological systems of increasing complexity but closely related to each other in a step-by-step sequence, e.g., bacterial mutagenesis; mammalian cell mutagenesis, toxicity and neoplastic transformation, including embryo cells, fibroblasts and epithelial cells; organ cultures of target epithelia; in vivo animal systems for short-term and long-term studies, including animal models closely comparable to human pathology; observational studies of human pathology and histopathogenesis; experimental studies of corresponding human target tissues using organ and cell culture methods for metabolism, toxicity, mutagenicity and possibly neoplastic cell transformation. Respiratory carcinogenesis models were successfully used for studies of mixed exposures to different carcinogens and cofactors. The role of particulates has been found to be important but needs to be further characterized. Quantitative variations in the response to carcinogens and cofactors among different biological test systems and among different individuals in the human population make quantitative risk estimation very difficult, but studies in a sequence of related biological systems including human tissues indicate the importance of qualitative risk evaluation.