In vitro human skin penetration of diethanolamine.

Concerns about the safety of diethanolamine (DEA) have been raised by the National Toxicology Program (NTP). Therefore, we measured the extent of DEA absorption in human skin relevant to exposures from shampoos, hair dyes and body lotions. Radiolabeled [14C]-DEA was added to two commercial products from each class and applied to excised viable and non-viable human skin in flow-through diffusion cells. The products remained on the skin for 5, 30 and 24 h for shampoos, hair dyes and body lotions, respectively. After 24 h, most of the absorbed dose was found in skin: 2.8% for shampoos, 2.9% for hair dyes and 10.0% for body lotions. Only small amounts were absorbed into the receptor fluid: 0.08%, 0.09% and 0.9% for shampoos, hair dyes and body lotions respectively. There was no significant difference in the absorption of DEA through viable and non-viable skin or from product application doses of 1, 2 or 3 mg lotion/cm2. In 72 h daily repeat dose studies with a lotion, DEA appeared to accumulate in the skin (29.2%) with little diffusing out into the receptor fluid. Therefore, skin levels of DEA should not be included in estimates of systemic absorption used in exposure assessments.

Percutaneous penetration and metabolism of 2-nitro-p-phenylenediamine in human and fuzzy rat skin.

2-Nitro-p-phenylenediamine (2NPPD) is a dye used in semipermanent and permanent (tinting color) hair dye formulations. National Toxicology Program toxicology and carcinogenesis testing of 2NPPD has raised concerns about its safety. Therefore, we initiated in vitro studies to measure absorption and metabolism of 2NPPD in human and fuzzy rat skin and rat jejunal tissue. Intestinal tissue metabolism of 2NPPD was compared to skin metabolism since toxicology data from oral 2NPPD studies will be used for future safety assessment purposes. Absorption was measured over 24 h by using flow-through diffusion cells with a receptor fluid consisting of Hepes-buffered Hank's balanced salt solution. Dosing vehicles were applied to skin and intestine in the diffusion cells for 30 min. 2NPPD metabolites were determined by high-performance liquid chromatography methodology. In human skin, the percentages of total applied dose absorbed (receptor fluid + skin) over 24 h were 9.2 +/- 5.7 (mean +/- SD) and 9.5 +/- 3.2 for the ethanol and semipermanent vehicles, respectively, with approximately 3% remaining in skin. In rat skin, the percentages of total applied dose absorbed over 24 h were 9.3 +/- 1.2 (mean +/- SE), 6.9 +/- 1.2, and 4.2 +/- 0.1 for the ethanol, semipermanent, and permanent formulation vehicles, respectively, with approximately 3% remaining in skin. In rat intestinal tissue, the percentage of total applied dose absorbed over 24 h was 10.9 +/- 1.2, with approximately 5% remaining in the tissue. In human and rat skin, 2NPPD was metabolized to triaminobenzene and N4-acetyl-2NPPD. 2NPPD was also metabolized to a sulfated 2NPPD metabolite in rat skin, but not in human skin. 2NPPD was extensively metabolized in both human and rat skin with ethanol application; metabolism was not as extensive with a semipermanent formulation application. In rat intestinal tissue, 62% of 2NPPD was metabolized upon absorption to triaminobenzene and N4-acetyl-2NPPD. Differences in the metabolic profiles (proportion of each metabolite formed) were found between the skin and intestinal tissue. These results suggest that 2NPPD is rapidly absorbed and extensively metabolized in both skin and intestinal tissue. The extent of metabolism and the metabolic profile were found to be species-, tissue-, and dosing vehicle-dependent. Metabolism information will be useful in predicting the extent of 2NPPD and/or 2NPPD metabolite systemic absorption relative to a dermal exposure, which will improve the health hazard assessment of 2NPPD.

Percutaneous absorption and metabolism of Coumarin in human and rat skin.

Coumarin is widely used as a fragrance in cosmetics, perfumes and soaps. The food and Drug Administration banned coumarin use in food because of reports that coumarin produced hepatotoxicity in rodents. Concerns about coumarin's safety have also been raised by toxicity testing conducted by the National Toxicology Program. Therefore, we initiated studies to measure the extent of coumarin absorption and metabolism in skin. [14C]Coumarin (ca. 0.5 microCi per cell) absorption in skin was measured by using two vehicles: ethanol (15 microliters cm-2) and an oil-in-water emulsion (3 mg cm-2). Absorption was determined for 24 h by using flow-through diffusion cells (0.64 cm2, exposed skin) with a receptor fluid consisting of HEPES-buffered Hank's balanced salt solution (pH 7.4). Coumarin metabolism was determined by high-performance liquid chromatography methodology. In rat skin (n = 3), the percentages of applied dose absorbed after 24 h were 54.9 +/- 0.63 (mean +/- SEM) and 86.8 +/- 5.4 for the ethanol and emulsion vehicles, respectively, with ca. 5% remaining in skin. In human skin (n = 2), the percentages of applied dose absorbed after 24 h were 64.4 +/- 0.29 and 98.0 +/- 5.3 for the ethanol and emulsion vehicles, respectively, with ca. 1% remaining in skin. The extent of skin absorption was greater from the emulsion vehicle than from the ethanol vehicle in both human and rat skin. Coumarin rapidly penetrated both rat and human skin with > 75% and > 95%, respectively, of the absorbed dose found in the receptor fluid within 6 h. No evidence of coumarin metabolism was found in either skin or receptor fluid fractions. These studies indicate that coumarin absorption is significant in skin. Systemic coumarin absorption must be expected after dermal contact with coumarin-containing products.

Reduction of erythema in hairless guinea pigs after cutaneous sulfur mustard vapor exposure by pretreatment with niacinamide, promethazine and indomethacin.

Erythema is the initial symptom that occurs after sulfur mustard (HD) cutaneous exposure. The time course of HD-induced erythema is similar to that observed after UV irradiation, which can be reduced by indomethacin. Sulfur mustard lethality is decreased by using promethazine, which is an antihistamine. Niacinamide can reduce microvesication after HD vapor exposure in hairless guinea pig (HGP) skin. The present study examines the effect of the combined administration of niacinamide, indomethacin and promethazine used alone or in all possible combinations on the degree of erythema and histopathologic skin damage after HD exposure in HGP. Niacinamide (750 mg kg-1, i.p.), promethazine (12.5 mg kg-1, i.m.) or indomethacin (4 mg kg-1, p.o.) used singly or in combination was given as a 30-min pretreatment before an 8-min HD vapor cup skin exposure. Using a combination pretreatment of niacinamide, promethazine and indomethacin, erythema was reduced at 4 (91%) and 6 (55%) h, but not 24 h after HD. The incidence of histopathological skin changes (microvesicles, follicular involvement, epidermal necrosis, intracellular edema and pustular epidermatitis) 24 h after HD was not reduced. This study indicates that HD-induced erythema may result from several different mechanisms, including inflammation, histamine release and DNA damage. It is suggested that two phases of inflammation may occur: an early phase sensitive to antihistamines and non-steroidal antiinflammatory drugs and a late phase of extensive cell damage that was not sensitive to these drug pretreatments.

Pathogenesis of 2,2'-dichlorodiethyl sulfide in hairless guinea pigs.

Developing skin lesions on hairless guinea pigs due to 2,2'-dichlorodiethyl sulfide (sulfur mustard, HD) exposure were examined to determine the time course for the appearance of histopathologic markers in relationship to skin NAD+ and NADP+ content after HD exposure. Hairless guinea pig skin was exposed to HD for 8 min by means of a vapor cup. Skin punches were taken at 1, 2, 4, 8, 12, 16, 20 and 24 h after HD exposure. Intracellular edema (IE) appeared at 2 h and increased steadily over 24 h. Epidermal necrosis (EN) and pustular epidermatitis (PE) developed at 8 h and reached a maximum at 16 h. Follicular necrosis (FN) appeared at 8 h and increased up to 24 h. Microvesicles (MV) developed between 12-16 h reaching a maximum at 24 h. Niacinamide (750 mg/kg, ip) pretreatment (30-min) reduced the incidence of MV (40%) and FN (45%) at 24 h, but did not reduce IE, EN, or PE. In all animals, skin NAD+ content decreased to a minimum (20% of control) at 16 h, but NAD+ decreases did not precede microvesicle formation. Skin NADP+ content increased (260%) between 1-2 h and returned to control at 4 h. Skin cell NADP+ increases may be indicative of an early phase of cellular oxidative stress that may contribute to HD-induced dermal pathogenesis. Since NAD+ reductions did not precede microvesication and NAM-induced increases in NAD+ content did not delay or reduce early cellular alterations, the contributory role of NAD+ to microvesicle formation may be limited and other biochemical changes should be investigated.

Sulfur mustard-increased proteolysis following in vitro and in vivo exposures.

The pathologic mechanisms underlying sulfur mustard (HD)-induced skin vesication are as yet undefined. Papirmeister et al. (1985) postulate enhanced proteolytic activity as a proximate cause of HD-induced cutaneous injury. Using a chromogenic peptide substrate assay, we previously reported that in vitro exposure of cell cultures to HD enhances proteolytic activity. We have continued our investigation of HD-increased proteolytic activity in vitro and have expanded our studies to include an in vivo animal model for HD exposure. In vitro exposure of human peripheral blood lymphocytes (PBL) to HD demonstrated that the increase in proteolytic activity is both time- and temperature-dependent. Using a panel of 10 protease substrates, we established that the HD-increased proteolysis was markedly different from that generated by plasminogen activator. The hairless guinea pig is an animal model used for the study of HD-induced dermal pathology. When control and HD-exposed PBL and hairless guinea pig skin where examined, similarities in their protease substrate reactivities were observed. HD-exposed hairless guinea pig skin biopsies demonstrated increased proteolytic activity that was time-dependent. The HD-increased proteolytic response was similar in both in vitro and in vivo studies and may be useful for elucidating both the mechanism of HD-induced vesication and potential treatment compounds.

Sulfur mustard-induced microvesication in hairless guinea pigs: effect of short-term niacinamide administration.

It has been postulated that sulfur mustard (HD) damage may activate poly(ADP-ribose) polymerase (PADPRP), resulting in depletion of cellular NAD+. This biochemical alteration is postulated to result in blister (vesicle) formation. It has been previously demonstrated that niacinamide (NAM), an inhibitor of PADPRP and a precursor for NAD+ synthesis, may be useful as a pretreatment compound to reduce HD-induced microvesication. The present study was undertaken to determine whether niacinamide's protective action could be extended beyond 24 hr and if the degree of microvesication is related to changes in skin NAD+ content. HD exposures were made by vapor cup to hairless guinea pigs. Niacinamide (750 mg/kg, ip) given as a 30-min pretreatment did not reduce the degree of microvesication 72 hr after HD compared to saline controls. However, niacinamide given as a 30-min pretreatment and at 6-, 24-, and 48-hr after HD, exhibited a 28% reduction in microvesication 72 hr after HD. Skin NAD+ content at 72 hr after HD was depleted by approximately 53% in the saline and NAM-treated groups. Skin NAD+ content was depleted despite NAM administration. Niacinamide did not reduce the degree of erythema at 48 or 72 hr. These results suggest that niacinamide's protective effect against HD-induced microvesication may be extended for at least 72 hr, but NAM levels must be sustained during the post-HD period. The link between maintenance of skin NAD+ and reductions in microvesication is still uncertain.

Monoclonal antibodies against soman: characterization of soman stereoisomers.

Hybridomas were produced which expressed monoclonal anti-soman antibodies as determined by microtiter enzyme-linked-antibody immunoassay (EIA). Each of these antibodies was titrated using a competitive inhibition enzyme immunoassay (CIEIA) with a variety of test ligands. The ligands used included soman (a racemic mixture), sarin, tabun, and each of the four stereoisomers of soman (C+ P+, C+ P-, C-P+ and C-P-). In all cases the antibodies tested exhibited IC50 values of 10(-4)-5 x 10(-6) M for soman. When sarin or tabun was used as a ligand, the antibodies exhibited no cross reactivity. All of the antibodies cross reacted with the four soman stereoisomers. A second group of hybridomas were produced which expressed monoclonal antibodies against CsPs-soman. These antibodies were used to make preliminary absolute chiral assignments to the four soman stereoisomers.

Niacinamide pretreatment reduces microvesicle formation in hairless guinea pigs cutaneously exposed to sulfur mustard.

It has been proposed that sulfur mustard (HD) may indirectly activate poly(ADP-ribose) polymerase (PADPRP) by alkylating cellular DNA (Papirmeister et al., 1985). Activation of PADPRP results in the depletion of cellular NAD+, which initiates a series of biochemical processes that have been proposed to culminate in blister formation. Preventing PADPRP activation and NAD+ depletion should inhibit blister formation. Niacinamide is both an inhibitor of PADPRP and a precursor for NAD+ synthesis. The present study was undertaken to determine whether niacinamide can protect against HD-induced microvesication in cutaneously exposed hairless guinea pigs. Each site was exposed to HD for 8 min by means of a vapor cup. Niacinamide (750 mg/kg, ip) given as a 30-min pretreatment inhibited microvesicle formation by 50% after HD application. However, niacinamide given 2 hr after HD application did not reduce microvesicle formation. There was no benefit when niacinamide was given as both a pretreatment and treatment when compared to niacinamide given only as a pretreatment. The reduction in microvesication 24 hr after HD did not correlate with skin NAD+ content. Niacinamide did not reduce the degree of erythema or edema. Ballooning degeneration of basal epidermal cells was present in some niacinamide pretreated HD exposure sites. These results suggest that niacinamide may only be effective as a pretreatment compound to reduce the incidence of HD-induced microvesicle formation. Maintenance of skin NAD+ content may not be solely responsible for inhibiting microvesicle formation and inhibition of PADPRP may be of greater importance.

Disulfiram metabolism as a requirement for the inhibition of rat liver mitochondrial low Km aldehyde dehydrogenase.

In humans and animals, disulfiram produces a disulfiram-ethanol reaction after an ethanol challenge, the basis of which is the inhibition of liver aldehyde dehydrogenase (ALDH). Disulfiram and the metabolites diethyldithiocarbamate (DDTC), diethyldithiocarbamate-methyl ester (DDTC-Me), and S-methyl-N,N-diethylthiolcarbamate (DETC-Me) were studied in order to determine the role of bioactivation in disulfiram's action as an inhibitor of rat liver mitochondrial low Km ALDH (RLM low Km ALDH). In in vitro studies, disulfiram and DDTC (0.01 to 2.0 mM) both inhibited RLM low Km ALDH in a concentration-dependent manner. The addition of rat liver microsomes to the mitochondrial incubation did not further increase disulfiram-induced RLM low Km ALDH inhibition. However, DDTC-induced RLM low Km ALDH inhibition was increased further, but only at DDTC concentrations less than 0.05 mM. DDTC-Me and DETC-Me (2.0 mM) similarly exhibited an increased RLM low Km ALDH inhibition after the addition of liver microsomes. In in vivo studies, disulfiram (75 mg/kg), DDTC (114 mg/kg), DDTC-Me (41.2 mg/kg) or DETC-Me (18.6 mg/kg) administered i.p. to female rats inhibited RLM low Km ALDH. Inhibition of drug metabolism by pretreatment of rats with the cytochrome P450 inhibitor N-octylimidazole (NOI) (20 mg/kg, i.p.) prior to either disulfiram, DDTC, DDTC-Me or DETC-Me administration blocked the inhibition of RLM low Km ALDH. The in vitro and in vivo data support the conclusion that bioactivation of disulfiram to a reactive chemical species is required for RLM low Km ALDH inhibition and a disulfiram-ethanol reaction.

Regeneration of acetylcholinesterase in clonal neuroblastoma-glioma hybrid NG108-15 cells after soman inhibition: effect of glycyl-L-glutamine.

Acetylcholinesterase (AChE) in the clonal NG108-15 cell line has been previously characterized. This cell line represents an in vitro system to study AChE regulation and effects of chemical compounds that may alter AChE activity. Recently, glycyl-L-glutamine (GLG) was demonstrated to function as a neurotrophic factor for maintenance of AChE content in cat denervated superior cervical ganglion cells. In the present study, regeneration of AChE activity in cultures of undifferentiated NG108-15 cells after soman inhibition was investigated in the presence and absence of GLG. Cells were treated with soman (5.5 x 10(-6) M) for 15 min and then washed to remove excess soman. Culture medium containing either GLG (10(-6), 10(-5), or 10(-4) M) or glycyl-L-glutamic acid (10(-6) M) was added to cultures after soman treatment and remained in the medium until cell harvest. Cells were physically detached at various times after soman treatment and specific AChE activity was determined. After soman, AChE activity dramatically decreased to less than 1% of untreated cellular activity at 1 hr. AChE activity gradually increased after 5 hr, while untreated cell AChE activity was regained 20 hr after soman. The t1/2 for AChE regeneration was approximately 10 hr. GLG did not increase the rate of AChE regeneration after soman inhibition. These results indicate that GLG is not a directly acting neurotrophic factor for AChE synthesis in NG108-15 cells after chemical AChE inactivation.

S-methyl-N,N-diethylthiolcarbamate: a disulfiram metabolite and potent rat liver mitochondrial low Km aldehyde dehydrogenase inhibitor.

S-methyl-N,N-diethylthiolcarbamate-methyl ester (DETC-Me), a proposed disulfiram metabolite, was investigated both in vivo and in vitro for its effectiveness as a liver mitochondrial low Km aldehyde dehydrogenase (L Km ALDH) inhibitor. Male Sprague-Dawley rats were treated intraperitoneally with DETC-Me, killed at various times and L Km ALDH determined. DETC-Me was found to be a more potent in vivo inhibitor of L Km ALDH than either disulfiram, diethyldithiocarbamate (DDTC) or diethyldithiocarbamate-methyl ester (DDTC-Me). The ID50 for DETC-Me, DDTC-Me and disulfiram was 6.5, 15.5 and 56.2 mg/kg, respectively. The ID50 for DDTC was similar to DDTC-Me. Maximal inhibition of L Km ALDH occurred 30 minutes after DETC-Me administration. DETC-Me was ineffective as an in vitro inhibitor. DETC-Me produced a marked disulfiram-ethanol reaction (DER) at one-quarter of the dose of disulfiram or DDTC. Plasma DETC-Me in rats was greater after DETC-Me administration than after DDTC-Me, DDTC or disulfiram. In conclusion, DETC-Me is proposed to be a metabolite of disulfiram, and may be the immediate precursor of the chemical species responsible for L Km ALDH inhibition.

Comparative aspects of disulfiram and its metabolites in the disulfiram-ethanol reaction in the rat.

Diethyldithiocarbamate-methyl ester (DDTC-Me), a metabolite of disulfiram, has been shown recently to produce a disulfiram-ethanol reaction (DER). Studies were carried out to compare the ethanol-sensitizing properties of DDTC-Me with those of disulfiram and diethyldithiocarbamate (DDTC) in the rat. All three drugs inhibited liver mitochondrial low Km aldehyde dehydrogenase (ALDH) in vivo, with maximal ALDH inhibition occurring 8 hr after drug administration. The onset of ALDH inhibition was most rapid after DDTC-Me administration. ALDH was inhibited approximately 50% 0.5 hr after DDTC-Me, whereas ALDH was inhibited only 5 and 10%, respectively, after disulfiram and DDTC. Not until 8 hr after drug treatment was ALDH inhibition the same for disulfiram, DDTC and DDTC-Me. The degree of ALDH inhibition from 8 to 172 hr after dosing was the same for all three drugs. An ethanol (1 g/kg, 20% v/v) challenge administered to rats treated with disulfiram (75 mg/kg), DDTC (114 mg/kg), or DDTC-Me (41.2 mg/kg) for 8 hr produced similar blood acetaldehyde/ethanol concentration-time profiles. In addition, all three agents produced a DER (hypotension, tachycardia). No DER occurred if ethanol was administered more than 24 hr after drug pretreatment. The hypotension associated with the DER correlated with the increased blood acetaldehyde but not blood ethanol. A threshold blood acetaldehyde of 110 microM appeared to be required for hypotension to occur, and this was related to ALDH inhibition of approximately 40%. The tachycardia associated with the DER correlated more with blood ethanol. After DDTC-Me administration, no disulfiram or DDTC could be detected in the plasma. Furthermore, no DDTC-Me was found in the plasma 8 hr after DDTC-Me administration, suggesting that no correlation exists between the DER and plasma concentration of DDTC-Me and most likely disulfiram. These data suggest that the alcohol-sensitizing properties of DDTC-Me are similar to those observed with disulfiram and DDTC. Since DDTC-Me is an active metabolite and more potent than disulfiram and DDTC in producing a DER, disulfiram metabolism is an important consideration in the disulfiram-ethanol reaction.

Diethyldithiocarbamic acid-methyl ester: a metabolite of disulfiram and its alcohol sensitizing properties in the disulfiram-ethanol reaction.

Diethyldithiocarbamic-acid-methyl ester (DDTC-Me) is a major metabolite of disulfiram. When given to rats, DDTC-Me was found to inhibit the liver mitochondrial low Km aldehyde dehydrogenase (ALDH) without having any effect on the high Km isoenzyme. Inhibition of low Km ALDH by DDTC-Me in vivo exhibited a dose-response relationship, with inhibition of ALDH from 11% to 90% found when DDTC-Me was administered in a dose range from 1.8 to 158 mg/kg, IP. After a single dose of DDTC-Me (41.2 mg/kg, IP), the low Km ALDH was inhibited for 168 hours suggesting an irreversible enzyme inhibition. After an ethanol challenge to DDTC-Me-treated rats, a decrease in mean arterial pressure (MAP) and increase in heart rate was observed. Decreases in MAP occurred almost immediately after ethanol challenge and remained low throughout a four hour post-ethanol period. These results suggest that in vivo administration of DDTC-Me can cause an alcohol-sensitizing reaction, and that DDTC-Me actually may be the metabolite of disulfiram which produces the disulfiram-ethanol reaction. It is proposed the reaction be more correctly identified as the DDTC-Me-Ethanol Reaction or D-MER.

The effects of insulin on hepatic glucocorticoid receptor content in the diabetic rat.

Streptozotocin-induced diabetic rat liver was analyzed for glucocorticoid receptor (GR) content by saturation and Scatchard analysis. The hepatic GR content of streptozotocin-induced diabetic rats was significantly decreased from a control level of 0.17 +/- .01 pmol/mg protein to 0.11 +/- .01 pmol/mg protein. Insulin replacement therapy to the diabetic rat dramatically increased the hepatic GR content to 0.26 +/- 0.02 pmol/mg protein as compared to the diabetic value of 0.11 +/- 0.01 pmol/mg protein. A time course study of GR content in the diabetic rat liver demonstrated that after an initial decrease in hepatic GR content at 14 days, the 25-day diabetic receptor level elevated back to control levels. A significant increase in GR content over controls was observed in the 110-day diabetic rats. These results suggest that insulin has a role in the regulation of hepatic GR content.