The objective of this study was to determine the absorption and disposition in rats of two cobalt compounds: cobalt chloride, an aqueous-soluble compound, and cobalt naphthenate, an aqueous-insoluble compound. Their in vitro dissolution rates were investigated to determine whether they correlate with their toxicity. Dissolution of cobalt naphthenate was strongly media and pH dependent and showed increased dissolution in the presence of protein and at low pH. Cobalt oxide, an aqueous-insoluble cobalt compound, was unaffected by solution conditions. The in vitro alveolar macrophage results indicated that compounds added as particulates were more cytotoxic than those added in solution. Cobalt chloride was the most cytotoxic and cobalt oxide the least. These results suggest that the order of in vitro cytotoxicity to alveolar macrophages may be predicted from their in vitro dissolution behavior. When the in vitro dissolution of the cobalt compounds was tested at a pH of 2 to model the environment inside the stomach, the dissolution of cobalt naphthenate and cobalt chloride was identical. This indicated that oral exposure to cobalt naphthenate could result in essentially complete dissociation of cobalt at gastric pH. The distribution and elimination of cobalt naphthenate was identical to that of an equivalent Co(II) dose of cobalt chloride. The oral blood cobalt concentration curves were triphasic and exhibited similar pharmacokinetic parameters. Following intravenous administration, approximately 10% of the dose was found in the feces, indicating that cobalt can be secreted in the bile. The intravenous cobalt concentration curve was also triphasic with a terminal elimination half-life of 19.0 hr. Intestinal ring incubation experiments indicated that cobalt transport has both active and passive components. The finding that uptake was saturable may explain the small degree of absorption following oral dosing. The results of the heme oxygenase assays indicated that subcutaneous and intravenous administration resulted in increased activity over controls at an equivalent hepatic Co(II) content. Thus, these results demonstrated that the extent of cobalt absorption across the gastrointestinal tract is incomplete and that concentration and route of exposure may determine its systemic toxicity.

1,2-Dichlorobenzene (1,2-DCB) is a potent hepatotoxicant in the Fischer-344 (F344) rat. Phenobarbital (PB), a known inducer of cytochrome P-450, enhanced the metabolism and covalent binding of 1,2-DCB in F344 rat liver microsomes. Identification of 2,3-dichlorophenol, 3,4-dichlorophenol and dichlorobenzene dihydrodiol indicated the formation of dichlorobenzene epoxides in PB induced microsomes. Moreover, modulation of microsomal metabolism and covalent binding using glutathione, trichloropropene oxide, ascorbic acid and superoxide dismutase implicated quinones as the major covalent binding species of 1,2-DCB. These findings indicate that 1,2-DCB is activated by cytochrome P-450 to reactive intermediates that may initiate hepatocellular injury. The hepatotoxicity of 1,2-DCB was also studied in normal F344 rats administered methyl palmitate (MP) to inhibit Kupffer cell function or superoxide dismutase (conjugated to polyethylene glycol, i.e. PEG-SOD) to scavenge superoxide anions. Both agents markedly reduced the severity of 1,2-DCB induced liver injury in normal rats. However, MP and PEG-SOD did not inhibit the PB potentiated hepatotoxicity of 1,2-DCB. In summary, the data presented in this dissertation strongly support a role for Kupffer cell derived superoxide anions in the hepatotoxicity of 1,2-DCB in normal F344 rats. Since Sprague-Dawley (SD) rats are less susceptible to the hepatotoxicity of 1,2-DCB than F344 rats, markers of oxidant injury were assessed in both strains of rats following administration of 1,2-DCB. 1,2-DCB treatment did not deplete hepatic vitamin E in F344 or SD rats. However, 1,2-DCB treated F344 rats exhibited greater ethane exhalation than SD rats, at a time when differences in GSH depletion between the two strains were most prominent and prior to the initial appearance of toxicity in F344 rats. The results further confirmed the involvement of oxidative injury in the hepatotoxicity of 1,2-DCB. It is concluded that two key events are involved in the hepatotoxicity of 1,2-DCB (1) metabolism of 1,2-DCB by cytochrome P-450 to reactive intermediates that initiate cell injury (2) oxidative injury induced by Kupffer cell derived active oxygen species, that contributes to the progression of the injury.

The volatile anesthetic halothane can be bioactivated in the liver to the reactive intermediate, trifluoroacetyl chloride, which is capable of covalently modifying liver protein. The product of this reaction is trifluoroacetyl-N-ε-amino lysine, which can act as a foreign epitope in altering both protein immunogenicity and antigenicity. Protein adduct formation appears to be responsible for the development of both an acute and an immune-mediated hepatotoxicity. The goal of this research project was to detect, quantify, and characterize the formation of protein adducts in the guinea pig liver, following exposure to halothane. This species provides the most accurate animal model for halothane hepatitis to date. An in vitro liver slice system was used to study the conditions for the production of protein adducts during halothane exposure. Covalent binding to slice protein occurred in a linear fashion over the time course of exposure, and was concentration dependent. Oxidative metabolism of halothane was required for adduct production. Adduct formation occurred to specific and identifiable proteins. The majority of the protein adducts in the liver slice were localized to cytosolic glutathione-S-transferase (GST). GST can be released from the liver slice, transporting the adduct to the extracellular environment. Guinea pigs were anesthetized with halothane to compare the results obtained in vitro, with what occurs in the whole animal. Covalent binding to liver protein occurred predominately in the microsomal fraction. The protein adducts identified in the guinea pigs corresponded to those seen in liver slices. GST was identified as a target for the acid chloride intermediate in the liver of these animals. Covalent binding to cytosolic protein was dependent on liver glutathione content. A specific relationship between adduct formation to cytosolic protein and glutathione concentration was further demonstrated using an in vitro bioactivation system. GST may catalyze the reaction between the electrophile and glutathione. Liver glutathione content appears to mediate the degree and selectivity of covalent binding to target proteins. The development of halothane induced hepatotoxicity may be related to the interactions between its reactive intermediate, glutathione, and GST.

The mechanism by which vitamin A (VA, retinol) potentiates the hepatotoxicity of carbon tetrachloride (CCl₄) in male Srague-Dawley rats was investigated. The toxicity of single and repeated doses of CCl₄ was potentiated in rats following VA treatment. CCl₄-induced hepatotoxicity was completely eliminated in control and VA-treated rats by 1-aminobenzotriazole, a cytochrome P-450 inhibitor, indicating that CCl₄ metabolism was necessary to achieve potentiation. To determine if VA-potentiated CCl₄ hepatotoxicity involves retinol activation of Kupffer cells (KC), various parameters were measured as indicators of KC function. In vitro assays using populations of KC demonstrated that phagocytosis and free radical release were increased in KC isolated from VA-treated rats. A novel electrooptical technique for measuring release of superoxide anion (•O₂-) from individual KC was developed. It was demonstrated that KC are a heterogeneous population, each responding separately to a common stimulus. To examine if the mechanism by which VA potentiates CCl₄ hepatotoxicity is common to model systems in addition to Sprague-Dawley rats, four separate mice strains were also tested. VA pretreatment dramatically protected against CCl₄ toxicity in each mouse strain tested. These data support the hypothesis that in rats, release of reactive oxygen intermediates from KC is intimately involved in the mechanism by which VA potentiates CCl₄ hepatotoxicity.

The development of a clinical diagnostic scheme for ciguatera fish poisoning has been accomplished through the utilization of three assays, specifically a competitive enzyme-linked immunosorbent assay (ELISA) for quantification, and two procedures for separation of toxic potentials, i.e., a liquid-liquid partition method and an immunoaffinity column system. For the ELISA assay, quality control parameters were evaluated including the coating binding efficiency and the binding capability of the conjugate (okadaic acid-bovine serum albumin). A 100% recovery was obtained and reproducibility values were under 30% coefficient of variation (CV) for concentrations from 0.0001 to 100 ng okadaic acid/mL serum or g fish flesh. Additionally, the usefulness of the method was evaluated by determining sensitivity and specificity parameters (66.7 and 95%, respectively) from where the positive and negative predictive values of the test as a diagnostic instrument were assessed. A rapid extraction procedure utilizing a mixture of methanol:chloroform:water was adapted for extraction and isolation of toxins associated with ciguatera fish poisoning. The method has been optimized for the analytical evaluation of potentially ciguatoxic fish samples and human serum. Applications of the extraction procedure include the clinical diagnosis of ciguatera illness and, if leftover fish portions responsible for the intoxication event are available, analysis of the seafood. It can also be used as a tool for monitoring seafood products for food safety. Recovery values of the extraction method with and without an intermediary defatting step for spiked fish samples were of 105 and 86%, respectively. By attaching anti-okadaic acid monoclonal antibody onto an inert matrix (AH-Sepharose®), an immunoaffinity column specific to ciguatera-related toxins has been developed, also for serum analysis, but with the potential application in other body fluids. The binding efficiency was 82% and reproducibility values for spiked test portions at concentrations as low as 250 pg were below 10%.

Pyrrolizidine alkaloids are proposed to be metabolized in the liver to reactive pyrrole species, or dehydroalkaloids. These reactive pyrroles are hypothesized to be responsible for pyrrolizidine alkaloid toxicity. This dissertation research has established that dehydroalkaloids are, in fact, metabolites of pyrrolizidine alkaloids. It was first determined that dehydromonocrotaline is produced during hepatic microsomal metabolism of monocrotaline and that it has the ability to bind in vitro with a synthetic thiol-containing resin, Thiopropyl Sepharose 6B. Similarly, synthetic dehydromonocrotaline binds to this resin. Dehydromonocrotaline was identified as a pyrrolizidine alkaloid metabolite based upon its resin cleavage products. When resin-bound pyrrole, synthetic or microsomally generated, was cleaved in a buffered, ethanolic silver nitrate solution, O⁷-ethyl dehydroretronecine was the major product, supporting the suggestion that the pyrrole generated by hepatic microsomes is dehydromonocrotaline. This system was then used to determine the formation of dehydroalkaloids from other pyrrolizidine alkaloids. These other alkaloids--trichodesmine, retrorsine, senecionine and heliotrine--cause toxicity to the liver as well as to extrahepatic organs. Their metabolism in this system reveals that alkaloids which produce extrahepatic toxicity have an increased percentage of reactive metabolites formed by phenobarbital-induced hepatic microsomes. Therefore, this system in vitro can be a good predictor of alkaloids which may produce extrahepatic toxicity in vivo. Trichodesmine is a pyrrolizidine alkaloid that is unique in its neurotoxicity. It is structurally similar to monocrotaline, yet it varies widely in its toxicity. It was determined that trichodesmine is more toxic in the rat than monocrotaline as indexed by LD₅₀ values. The distribution of pyrrolic metabolites reveals that trichodesmine treatment results in brain pyrrole levels 4 times higher than monocrotaline, retrorsine, or control. Histopathologic investigation of trichodesmine-treated animals reveals severe neuronal death in the cerebral cortex. These results suggest that neurotoxicity observed with trichodesmine is a result of pyrrole metabolites reaching the brain, thus providing further evidence for the involvement of pyrrole metabolites in pyrrolizidine alkaloid-induced extrahepatic toxicity.

The efficacy and safety of ammoniation procedures for reducing aflatoxin hazards in corn were determined by exposing naturally incurred aflatoxin B₁ (AFB₁) contaminated corn (17, 354, and 7,500 ng AFB₁/g) to various ammoniation processes. Treatment with gaseous NH₃ or NH₄OH under appropriate conditions resulted in 93∼99% decrease in AFB₁ levels. No significant reversion of aflatoxin was observed (reversibility < 0.05%). Moisture content in corn and holding temperature were the crucial factors influencing the efficacy of ammoniation. ¹⁴C-AFB₁ spiked corn sample (1 μCi/kg, original AFB₁ = 7500 ng/g) was subjected to sequential isolation and separation procedures (CH₂Cl₂, and CH₃OH extraction, followed by acetic acid and NaOH treatment or Pronase E digestion) to determine the distribution of ammonia/aflatoxin reaction products. Approximately 12% of the added radioactivity was volatilized during ammoniation; 19 and 13% with CH₂Cl₂ and CH₃OH extraction, respectively; and 19% with acid-base treatment or Pronase digestion. The final matrix following either the acid-base treatment or Pronase digestion contained 37% of the added radioactivity. The above isolates were then tested using the Salmonella/microsomal mutagenicity assay (TA100). Purified AFB₁ showed positive response around 10 ng/plate. All isolates from ammonia treated and untreated aflatoxin-contaminated corn did not exhibit positive mutagenic potential. Further study provided the evidence of CH₂Cl₂ extractable "unknown interfering materials" in the corn which may bind with aflatoxin and/or block the mutagenic activity of aflatoxin. These substances were not separated from the aflatoxins by the TLC. Further studies are required using different sequential solvent fractionation processes and different procedures of the Ames test to evaluate this phenomenon. Male F-344 rats were fed diets containing ammonia-treated and untreated aflatoxin-contaminated and aflatoxin-free corn for 14 day period. Terminal liver tissues were analyzed for AFB₁-DNA adducts. No AFB₁-induced DNA adducts were detected in controls and animals fed diets containing 75 ng AFB₁/g and 20 ng AFB₁+ 237 ng ammonia/AFB₁ reaction products. These data confirm the efficacy and permanency of the ammonia process to reduce aflatoxin levels and evidence of safety through absence of AFB$\sb1$-DNA adducts in rat liver.

Prostaglandin E2 (PGE2) is a hormone derived from the metabolism of arachidonic acid whose functions include regulation of platelet aggregation, fever and smooth muscle contraction/relaxation. PGE2 mediates its physiological and pathophysiological effects through its binding to four G-protein coupled receptor subtypes, named EP1, EP2, EP3 and EP4. The EP3 prostanoid receptor is unique in that it has multiple isoforms generated by alternative mRNA splicing. These splice variants display differences in tissue expression, constitutive activity and regulation of signaling molecules. To date there are few reports identifying differential activities of EP3 receptor isoforms and their effects on gene regulation.We generated HEK 293 EBNA cell lines expressing the EP3-Ia, EP3-II, or EP3-III isoforms. After confirming the functional expression of each of these isoforms, we examined their activation of cellular signal transduction pathways.We found that each of these isoforms utilize distinct mechanisms to regulate ERK 1/2 phosphorylation and that these differences lead to unique regulation of the downstream effectors ELK-1 and AP-1. We also found MAPK dependent differences in regulation of cell proliferation. The EP3-III isoform increases cell proliferation in a MAPK dependent manner while the EP3-Ia dose dependently regulates cell proliferation via Gαi and not ERK 1/2. Activation of the EP3-II receptor had no effects on cell proliferation.To study differential gene regulation by these three EP3 receptor isoforms, we conducted microarray studies. Over 300 genes were differentially regulated by these isoforms. Quantitative real-time PCR analysis was used to validate 15 candidate genes. Five genes were chosen for further analysis of protein expression using immunoblotting, but only one of these, WT-1, was significantly increased following treatment with PGE2. WT-1, a transcription factor important for kidney and heart development, was strongly upregulated by PGE2 stimulation of the EP3-II receptor, but only weakly by the other isoforms.In conclusion, these studies show that the human EP3 prostanoid receptor isoforms are capable of distinct regulation of both signal transduction pathways and gene transcription. Elucidating the differential functions of EP3 receptor isoforms may allow for greater understanding of the diverse functions attributed to this receptor and their physiological functions.

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