Marc W. Fariss

Treatment of Chlordecone (Kepone) Toxicity with Cholestyramine — Results of a Controlled Clinical Trial

Industrial workers exposed to the organochlorine pesticide, chlordecone (Kepone), had signs of toxicity in several organs. The extent of toxicity was proportional to the levels of this chemical in the tissues. In 22 patients, chlordecone was eliminated slowly from blood (half time of 165 +/- 27 days--mean +/- S.E.M.) and fat (half time of 125 days, with a range of 97 to 177), chiefly in the stool. Output of chlordecone in bile was 10 to 20 times greater than in stool, suggesting that chlordecone is reabsorbed in the ntestine. Cholestyramine, an anion-exchange resin that binds chlordecone, increased its fecal excretion by seven times. In a five-month trial, cholestyramine significantly accelerated elimination of chlordecone from blood, with a half life of 80 +/- 4 days (S.E.M.) (P less than 0.005) and fat (half life of 64 days, with a range of 52 to 85) (P less than 0.05). Cholestyramine offers a practical means for detoxification of persons exposed to chlordecone and possibly to other lipophilic toxins.

Cadmium toxicity: unique cytoprotective properties of alpha tocopheryl succinate in hepatocytes

Rat hepatocyte suspensions were exposed to toxic concentrations of cadmium (Cd) in the presence and absence of unesterified alpha-tocopherol (T) or alpha-tocopheryl succinate (TS). The exogenous administration of TS completely protected hepatocytes from Cd-induced injury and lipid peroxidation. However, hepatocytes exposed to T were not protected from the toxic manifestations of cadmium even though this treatment resulted in a rapid marked accumulation of cellular T. The rate of cadmium uptake by hepatocytes was not significantly altered by exogenous TS or T treatment. These studies indicate that TS cytoprotection against Cd toxicity results not from alterations in Cd uptake or the accumulation of T but rather from the cellular presence of the intact TS molecule. The data also indicate that the depletion of cellular T is not the critical cellular event that is responsible for Cd-induced injury. Instead it appears that TS possess unique cytoprotective properties that intervene in the critical cellular events that lead to Cd toxicity. Thus, TS administration represents a promising new strategy for the mechanistic study and prevention of tissue damage resulting from Cd exposure.

Oxygen toxicity : unique cytoprotective properties of vitamin E succinate in hepatocytes

Freshly isolated rat hepatocytes suspensions were incubated under an atmosphere of 95% O2/5% CO2 or 95% air/5% CO2 for 10 h. Cell injury and death were observed between the 6th and 10th hour of incubation, only in 95% O2-treated hepatocytes. Oxygen-induced injury was preceded by marked lipid peroxidation and rapid depletion of cellular alpha tocopherol content. The exogenous administration of unesterified alpha tocopherol (T, 25 microM) resulted in a 20-fold increase in cellular T levels (4.2 nmol/10(6) cells) but failed to protect these hepatocytes from the toxic effects of oxygen. In contrast, hepatocytes incubated with 25 microM of the succinate ester of alpha tocopherol (TS) contained both TS (3.0 nmol/10(6) cells) and T (1.4 nmol/10(6) cells) and were completely protected from the toxic effects of oxygen, including the induction of lipid peroxidation. These findings suggest that TS cytoprotection results not from the cellular accumulation of T but rather, from cellular TS accumulation. The data also indicate that the depletion of cellular T is not the critical cellular event that is responsible for hyperoxia (reactive oxygen intermediate)-induced injury. Instead, it appears that TS possesses unique cytoprotective properties that intervene in the critical cellular events that lead to oxygen toxicity. Thus, vitamin E succinate and our hyperoxic hepatocyte preparation provide a promising new model system for the study and prevention of tissue damage resulting from the toxic effects of hyperoxia and reactive oxygen intermediates.

α-tocopheryl succinate protects hepatocytes from chemical-induced toxicity under physiological calcium conditions

Rat and canine hepatocyte suspensions were exposed to toxic concentrations of ethyl methanesulfonate (EMS) and ionophore A-23187 in the presence and absence of extracellular calcium (Ca2+) and alpha-tocopheryl succinate (alpha-TS). The exogenous administration of alpha-TS (25 microM) completely protected hepatocytes from chemically-induced toxicity when exposed to physiological free extracellular calcium concentrations (0.8-1.5 mM). Under these protective conditions the cellular accumulation of both alpha-TS (2.8 nmol/10(6) cells) and alpha-T (0.91 nmol/10(6) cells) were observed. Hepatocytes exposed to unesterified alpha-tocopherol (alpha-T, 25 microM) or alpha-tocopheryl acetate (alpha-TA, 25 microM), however, were not protected from the toxic effect of chemicals even though these treatments resulted in the marked accumulation of cellular alpha-T (2.65 nmol/10(6) cells) and alpha-TA (2.3 nmol/10(6) cells), respectively. Our findings suggest that the supplementation of endogenous stores of alpha-T or alpha-TA does not promote protection against chemical toxicity and that alpha-TS cytoprotection results not from the accumulation of alpha-T but rather from the cellular presence of the intact alpha-TS molecule. Thus alpha-TS appears to possess cytoprotective properties that differ from other vitamin E congeners.

THE ROLE OF PLASMA PROTEINS IN THE TRANSPORT AND DISTRIBUTION OF CHLORDECONE (KEPONE®) AND OTHER POLYHALOGENATED HYDROCARBONS*

It has been amply documented that the blood of humans or animals exposed to organochlorine pesticides or other polyhalogenated hydrocarbon chemicals2* contains readily detectable quantities of these highly lipophilic substances. The presence of exceedingly hydrophobic chemicals in such an aqueous medium as blood might be explained by their dissolution in blood lipids. In support of this idea, Morgan and Roan4 showed that the concentrations of DDT, DDE, and dieldrin in most tissues including blood are directly proportional to the tissues concentration of lipid. An additional factor which contributes to the level of polyhalogenated hydrocarbons in the blood is binding of these chemicals to plasma proteins. Moss and Hathaway demonstrated association of telodrin and dieldrin with albumin and a-globulin in the blood of rabbits and rats. Subsequent studies by others have confirmed that many organochlorine compounds are bound by plasma proteins, particularly plasma lipoprotein^.^ Skalsky and GuthrieIb1* reported that binding of DDT and dieldrin by lipoproteins and albumin involved slowly reversible, hydrophobic interactions. These observations are important because evidence is accumulating that protein binding may be a key factor in establishing the distribution in the body of at least some of these organic chemicals. For example, Eliason and Posner* demonstrated that transplacental transport of dieldrin to the fetus is dependent not only upon the concentration of maternal plasma proteins, but also upon the ontogenic development of specific fetal plasma proteins. Morrison19 showed that the concentration of DDT in cerebral spinal fluid is higher than would be expected from the total content of lipid, a finding which suggests that the pesticide may be bound to proteins in the spinal fluid. We have reported another example of dissociation between the distribution of lipid and that of an organochlorine pesticide in chemical workers exposed to large quantities of chlordecone(Kepone@)? The ratio of the concentration of chlordecone in adipose tissue compared to blood was seven to one, a value strikingly lower when compared with the distribution of other organochlorine pesticides as, for example, DDT, which has a ratio of concentration in fat to blood of 280 to 1.2 From the

Role of oxidative stress and MAPK signaling in reference moist smokeless tobacco-induced HOK-16B cell death.

The use of smokeless tobacco products is often associated with an oral injury at the site of repeated use. To further our understanding of this injury process, the effect of reference moist smokeless tobacco extract (STE) on cell death, oxidative stress, and MAPK signaling in a human oral keratinocyte cell line, HOK-16B, was investigated. STE caused dose-dependent cell death and reactive oxygen species (ROS) production within 30 min to 3h of exposure. This same insult enhanced the activity of ERK1/2, JNK1/2, p38 MAPK and ASK1, an upstream activator of JNK1/2 and p38 MAPK. Inhibition of JNK1/2 and to a lesser extent p38 MAPK, but not ERK1/2, suppressed STE-induced cell death. Pretreatment with antioxidants and an iron chelator, deferoxamine suppressed ROS production, ASK1, JNK1/2 and p38 MAPK activation, and reduced cell death after STE exposure. Interestingly, extracellular free iron levels in STE (29.4+/-0.5 microM) were significantly elevated as compared with cell culture medium (4.9+/-0.6 microM) and the addition of extracellular free iron (14, 30 or 70 microM) to HOK-16B cultures (without STE) caused dose-dependent cell death after 3h. Thus, acute exposure to STE leads to HOK-16B cell death in part through oxidative stress via activation of ASK1 and the JNK1/2 and p38 MAPK pathways.

Inhibition of cholinesterase activity by tetrahydroaminoacridine and the hemisuccinate esters of tocopherol and cholesterol

The anticholinesterase properties of tetrahydroaminoacridine (THA, Tacrine), alpha-tocopheryl hemisuccinate (TS), and cholesteryl hemisuccinate (CS), given alone and in combination, were examined in vitro. Results from these studies indicate that: [1] THA is a potent inhibitor of acetylcholinesterase (AChE, IC50 of 0.40 microM) and butyrylcholinesterase (BChE, IC50 of 0.10 microM) with greatest inhibitory activity towards BChE; [2] TS and CS are weak inhibitors of BChE (IC50 of 100 microM and 168 microM, respectively) but potent inhibitors of ACHE (IC50 of 1.73 microM and 0.79 microM, respectively); [3] both TS and CS treatment in combination with THA significantly increased THAs anticholinesterase activity. The percentage AChE inhibition observed with this combination was often significantly greater than the sum of the individual values (synergistic). The addition of 0.5 microM CS or TS to an ACHE preparation reduced THAs IC50 value from 0.40 microM or 0.18 microM, respectively [4]; inhibition of AChE by THA, TS and CS are mixed non-competitive while THA inhibition of BChE is mixed non-competitive and TS and CS inhibition of BChE are simple non-competitive; and [5] inhibition of cholinesterases by TS and CS occurs immediately (50 to 75%), during the first 30 min of incubation (25 to 50%) and is dependent on the anionic charged portion of the molecule. In conclusion, our experimental data indicate that TS and CS are potent inhibitors of AChE activity and significantly potentiate the anticholinesterase activity of THA. Such potent and synergistic inhibition of AChE suggest that TS or CS, alone and in combination with THA, may prove beneficial in the treatment of organophosphate poisoning and Alzheimers disease.

Reference moist smokeless tobacco-induced apoptosis in human monocytes/macrophages cell line MM6

Moist smokeless tobacco use is associated with various types of oral injury, including leukoplakia and dippers pouch, although the mechanism by which the injury is caused still remains unclear. One possible mechanism is that moist smokeless tobacco affects the inflammatory response. For example, a study by Johnson et al. demonstrated a reduction in the volume density of macrophages and increased inflammation and redness at the smokeless tobacco placement site when compared to non-placement site. The current study investigated the direct effect of reference moist smokeless tobacco extract (STE) exposure on the viability of MM6 monocyte/macrophage cell line. The exposure of MM6 cells to various concentrations of STE, led to a significant and dose-related decrease in cell viability. Furthermore, STE exposure resulted in an increase in Annexin V/PI positive cells, an increase in TUNEL-positive cells, and cleaved PARP staining all of which were inhibited by pre-incubation with a pan-caspase inhibitor, suggesting that the observed STE toxicity was due to the induction of apoptosis. Next, the role of various moist smokeless tobacco-derived components in STE-induced apoptosis of MM6 cells was investigated. Our findings suggest that STE-induced osmotic stress, but not exposure to nicotine, plays an important role in STE-induced apoptosis of MM6 cells. Together, these data show for the first time that STE exposure leads to the induction of apoptosis in human monocyte/macrophage cells, which appears to be induced in part, by reference STE-mediated osmotic stress.

Role of plasma membrane disruption in reference moist smokeless tobacco-induced cell death

An oral injury is thought to presage the development of mucosal lesions that are common in moist smokeless tobacco (MST) users. The abrasion or mechanical stress caused by direct contact of MST with the oral mucosa may contribute to this injury by causing transient disruptions in the cell membrane. In order to test this hypothesis, we developed an in vitro exposure system that directly exposes cells to reference MST on a rocking platform to simulate the abrasion that might be experienced in the oral cavity when using MST. Using this treatment paradigm, we monitored plasma membrane disruption as a measure of cell wounding caused by direct interaction of the tobacco material itself with monolayer cultures of Het-1A immortalized human esophageal cells as a potential contributor to the injury process. We found that a washed reference MST preparation, in which MST-associated chemicals were removed but the tobacco material retained, causes cell wounding as indicated by the uptake through plasma membrane disruptions of a fluorescent marker normally impermeable to the cell. Having established that non-chemical properties of MST cause cell wounding, subsequent experiments revealed that cell wounding during simultaneous exposure to an aqueous MST-extract result in greater than additive cell death when compared to treatment with washed MST or MST-extract alone. Furthermore, we found that the high levels of free calcium found in MST-extract appear to be playing an important role. Taken together, these results indicate that MST-induced oral injury may result from a combined interaction of physical disruption of the plasma membrane by the tobacco material itself and the adverse effects of MST chemical constituents, notably high levels of calcium, that gain entry to the cell by way of MST-induced cell wounding.

METABOLISM AND BILIARY EXCRETION OF CHLORDECONE (KEPONE) IN HUMANS

ABSTRACT Male chemical workers exposed to large quantities of the organochlorine pesticide, chlordecone, had high concentrations of the chemical in blood, fat and liver, and exhibited toxic manifestations affecting primarily the nervous system, liver, and testes. Unlike other pesticides of this class, chlordecone was excreted prominantly in bile. However, only 5-10% of the biliary chlordecone appeared in the stool, suggesting that chlordecone is reabsorbed in the intestine. Oral administration of cholestyramine, a non-absorbable ion exchange resin which binds chlordecone in vitro , increased fecal excretion of chlordecone and accelerated its disappearance from body tissues. This was accompanied by amelioration of clinical toxicity. Examination of human stool for pesticide metabolites revealed chlordecone alcohol, a stable, reduced form of chlordecone, in concentrations four-fold higher than chlordecone. Bioreduction of chlordecone to chlordecone alcohol is species-specific since rats treated with chlordecone produced negligible amounts of chlordecone alcohol. Treatment of human bile with β-glucuronidase released additional chlordecone (9%) and chlordecone alcohol (93%) as did hydrolysis with H2SO4 which released chlordecone (28%), but not chlordecone alcohol. We conclude that the fate of chlordecone in man involves its uptake by the liver, enzymatic reduction, conjugation with glucuronic acid, partial conversion to unidentified polar forms, and excretion of these metabolites into bile. Appropriate assessment of potential adverse human health consequences of chronic exposure to chlordecone in the environment should take into account interspecies differences in chlordecone metabolism.