Barbara W. Kemppainen

Effects of skin storage conditions and concentration of applied dose on [3H]T-2 toxin penetration through excised human and monkey skin

Penetration of [3H]T-2 toxin through excised human and monkey skin stored at -60 degrees C was faster than through human and monkey skin stored at 4 degrees C, respectively. The permeability of refrigerated human skin was 34% of the permeability of refrigerated monkey skin. Increasing the concentration of [3H]T-2 toxin applied to the refrigerated monkey skin increased the amount of [3H]T-2 toxin penetrating the skin and enhanced the efficiency of penetration. Metabolites of [3H]T-2 toxin were identified in the receptor fluid bathing the dermal side of the excised human and monkey skin.

Developmental Exposures of Male Rats to Soy Isoflavones Impact Leydig Cell Differentiation

Testicular Leydig cells, which are the predominant source of the male sex steroid hormone testosterone, express estrogen receptors (ESRs) and are subject to regulation by estrogen. Following ingestion, the two major isoflavones in soybeans, genistin and daidzin, are hydrolyzed by gut microflora to form genistein and daidzein, which have the capacity to bind ESRs and affect gene expression. Thus, the increasing use of soy-based products as nondairy sources of protein has raised concerns about the potential of these products to cause reproductive toxicity. In the present study, perinatal exposure of male rats to isoflavones induced proliferative activity in Leydig cells. Isoflavones have the capacity to act directly as mitogens in Leydig cells, because genistein treatment induced Leydig cell division in vitro. Genistein action regulating Leydig cell division involved ESRs, acting in concert with signaling molecules in the transduction pathway mediated by protein kinase B (AKT) and mitogen-activated protein kinase (MAPK). Enhanced proliferative activity in the prepubertal period increased Leydig cell numbers, which alleviated deficits in androgen biosynthesis and/or augmented serum and testicular testosterone concentrations in adulthood. Together, these observations indicate that the perinatal exposures of male rats to isoflavones affected Leydig cell differentiation, and they imply that including soy products in the diets of neonates has potential implications for testis function.

Sphingolipid levels in catfish consuming Fusarium moniliforme corn culture material containing fumonisins

Fumonisin, a secondary metabolite of Fusarium moniliforme, is frequently found in foods and feeds of humans and animals. Fumonisins are specific inhibitors of sphinganine (sphingosine) N-acyltransferase, a key enzyme in the pathway for de novo sphingolipid biosynthesis and reacylation of sphingosine derived from dietary sources or complex sphingolipid turnover. The objective of this study was to evaluate the effect of F. moniliforme toxins on sphingolipids in year-2 channel catfish. In a 12-week feeding trial, four groups of catfish per treatment were fed pelleted balanced diets containing F. moniliforme cultured corn. The fumonisin B1 (FB1) concentrations in diets were 0.3 (control), 2.5, 5, 10, 20, 40, 80 and 240 mg/kg. The free sphinganine to free sphingosine ratio was significantly (P < 0.05) elevated (with exception of brain) at 10, 20, 40 and 80 mg FB1 per kg diet in kidney, serum, liver and muscle, respectively. The increase in free sphingolipid ratios observed were found to be due to increases in the levels of free sphinganine in tissues. These results demonstrate that a mode of action of F. moniliforme toxins in catfish is similar to other species (ponies, pigs, rats), and is suggestive of fumonisin toxicity. It also demonstrated the potential diagnostic value of ratios of free sphingolipids in catfish.

In vitro penetration of tritium-labelled water (THO) and [3H]PbTx-3 (a red tide toxin) through monkey buccal mucosa and skin

The permeability coefficients (Kp) for tritium-labelled water (THO) were determined in human and monkey skin, and monkey buccal mucosa. Kp of human skin (0.47 x 10(-3) cm/h) correlated favorably with previous reports. Kp of hydrated monkey skin for THO (0.77 x 10(-3) cm/h) was not significantly different (P greater than 0.05) from Kp of hydrated human skin (0.88 x 10(-3) cm/h). Kp of monkey buccal mucosa for THO (6.15 x 10(-3) cm/h) was significantly greater than that for monkey skin. Penetration and disposition of [3H]PbTx-3 into layers of monkey buccal mucosa and skin was determined. [3H]PbTx-3 (5-7 microCi) dissolved in 2 ml of water was applied to epithelial/epidermal surface (2.8 cm2) at zero time. The relative percent dose recovered from the upper layers of buccal mucosa (epithelium) and skin (epidermis) varied, but at each time interval was less than 2.5% of the dose. At most of the time intervals (2-24 h), a larger percent of the dose was recovered from the inner layer of the buccal mucosa (lamina propria) than from the inner layer of skin (dermis). After 24 h, as much as 34 or 13% of the dose was recovered from lamina propria or dermis, respectively. At each time interval studied, less than 2% of dose of [3H]PbTx-3 penetrated into the receptor fluid which bathed the inner surfaces of the lamina propria or dermis. The results of this study demonstrate that monkey buccal mucosa is more permeable than skin to THO and PbTx-3.

Methods for in vitro skin absorption studies of a lipophilic toxin produced by red tide.

The penetration and distribution of [3H]PbTx-3 into pig skin was determined using in vivo and in vitro methods. The dose used in each topical study was 0.3-0.4 micrograms/cm2 skin, with dimethylsulfoxide as the vehicle. In the in vivo study, mean cutaneous absorption after 48 h (expressed as percentage of the dose) was 11.5% (n = 3). In the in vitro study, mean cutaneous absorption after 48 h was 1.6% (n = 12), when based on accumulation of radioactivity in receptor fluid, or 9.9% when based on receptor fluid and dermis. [3H]PbTx-3 readily penetrated through the epidermis into the dermis, reaching maximal dermal accumulation at 4 h (9.1% in vivo and 18% in vitro). At 24 h, the amount in the dermis decreased to 2.3% and 15% in vivo and in vitro, respectively and at 48 h the amount in the dermis decreased to 8.2% in vitro. These results demonstrate the important role of the dermis as a reservoir for a lipophilic compound in both in vivo and in vitro percutaneous absorption studies.

Comparison of in vitro and in vivo biological activity of mycotoxins

In vitro assays developed to screen the cytotoxic activity of chemicals in murine (NIH/3T3) and bovine (BE 12-6) embryonic cells were used to determine the concentrations (microgram/ml) of mycotoxins which caused 50% lethality (LC50). Embryonic cells were seeded in 96 well plates, cultured for 72 hr with dilutions of each individual and combinations of mycotoxins, and stained and counted. Verrucarin A and roridin A had the strongest cytotoxic activity, and ergotamine tartrate was least toxic. Furthermore, results correlated with published values of in vivo activity, indicating this assay can be used for acute toxicity screening of compounds.

In vitro percutaneous penetration and metabolism of [3H]T-2 toxin: Comparison of human, rabbit, guinea pig and rat

The purpose of this research was to determine which species of laboratory animal provided the best approximation of in vitro percutaneous penetration and metabolism of T-2 in humans. The [3H]T-2 which penetrated discs of skin after 48 hr (expressed as per cent of dose, 581 ng/cm2) was 1.0, 1.4, 2.8 and 9.7% for the human, rabbit, guinea pig and rat when the vehicle was methanol. The penetration was 29.2, 19.6, 51.9 and 52.6% for the human, rabbit, guinea pig and rat when the vehicle was DMSO. When 2 concentrations were compared, 79 ng/cm2 and 581 ng/cm2 (the vehicle was methanol), the higher dose caused a significant (P less than 0.05) increase in the per cent of dose which penetrated human and guinea pig skin. Metabolism was extensive in the human, rabbit, and rat, with the main metabolite being HT-2 toxin. Previous studies comparing human to monkey indicated penetration in these 2 species was different when methanol was the vehicle. This study indicates that the rabbit provides the best approximation of human skin, both in terms of penetration kinetics and metabolic activity.

Evaluation of Monkey Skin as a Model for in Vitro Percutaneous Penetration and Metabolism of [3H]T-2 Toxin in Human Skin

Discs of abdominal skin (obtained from humans and hybrid monkeys at autopsy) were mounted on diffusion cells. The epidermal surfaces were dosed with [3H]T-2 dissolved in dimethyl sulfoxide (DMSO). The rate of [3H]T-2 penetration (expressed as ng/cm2/hr) through human skin was 0.38 +/- 0.10 and 3.85 +/- 0.96 (means +/- 95% confidence limit) when dosed with 74 and 582 ng/cm2, respectively. [3H]T-2 penetrated through monkey skin at the rate of 0.37 +/- 0.14, 0.80 +/- 0.43, 4.13 +/- 1.71, and 6.55 +/- 3.45 when dosed with 70, 155, 555 and 1063 ng/cm2, respectively. Analysis of the receptor fluid bathing human skin revealed 15% of the radioactivity was associated with T-2, 71% with HT-2 toxin (HT-2), and 6.3% with an unknown metabolite more polar than HT-2. The radioactivity in the receptor fluid bathing monkey skin was associated with T-2 (87%) and HT-2 (1.0%). The results are consistent with the hypothesis that metabolism of T-2 occurred during penetration through the excised skin and did not occur in the receptor fluid due to enzymes leaching out of the skin. These findings indicate that excised monkey skin is a good model for T-2 penetration through human skin when DMSO is the vehicle, but that dermal metabolism of T-2 is different in these two species.

Comparison of penetration and metabolism of [3H]diacetoxyscirpenol, [3H]verrucarin a and [3H]T-2 toxin in skin

The purpose of this research was to determine the rate of cutaneous penetration and metabolism of [3H]diacetoxyscirpenol (DAS) and [3H]verrucarin A (VCA) and compare these values to previously determined values for [3H]T-2 toxin (T-2), to compare the cutaneous penetration and metabolism of DAS in human and guinea-pig skin, and to compare the effects of dose and of two vehicles, methanol and dimethylsulphoxide (DMSO), on penetration rates. DAS or VCA was applied to the epidermal surface of excised skin, and the receptor fluid bathing the dermal surface was sampled periodically for 48 hr. Whether the applied dose (581 ng/cm2) was dissolved in methanol or DMSO, the rate of penetration through human skin was lower for VCA than for DAS or T-2, the rates for the two latter compounds being similar at this dose. Metabolism of DAS occurred during penetration through excised human skin and did not occur in the receptor fluid as a result of enzymes leaching out of the skin. VCA appeared to be metabolized by human skin, but this conclusion is tentative because of the relative instability of this compound. DAS penetrated significantly (P less than 0.05) faster through excised guinea-pig skin than through human skin. Metabolism of DAS was greater in human skin than in guinea-pig skin. When compared with methanol, DMSO increased the penetration of DAS and VCA by factors of between 7 and 52. At the low dose (79 ng/cm2) DAS penetrated human and guinea-pig skin significantly (P less than 0.05) faster than T-2 using either vehicle.

Testicular Development in Male Rats Is Sensitive to a Soy-Based Diet in the Neonatal Period

ABSTRACT Approximately 30% of infants in the United States are exposed to high doses of isoflavones resulting from soy infant formula consumption. Soybeans contain the isoflavones genistin and daidzin, which are hydrolyzed in the gastrointestinal tract to their genistein and daidzein aglycones. Both aglycones possess hormonal activity and may interfere with male reproductive development. Testosterone, which supports male fertility, is mainly produced by testicular Leydig cells. Our previous studies indicated that perinatal exposure of male rats to isoflavones induced proliferative activity in Leydig cells and increased testosterone concentrations into adulthood. However, the relevance of the neonatal period as part of the perinatal window of isoflavone exposure remains to be established. The present study examined the effects of exposure to isoflavones on male offspring of dams maintained on a casein-based control or whole soybean diet in the neonatal period, that is, Days 2 to 21 postpartum. The results showed that the soybean diet stimulated proliferative activity in developing Leydig cells while suppressing their steroidogenic capacity in adulthood. In addition, isoflavone exposure decreased production of anti-Müllerian hormone by Sertoli cells. Similar to our previous in vitro studies of genistein action in Leydig cells, daidzein induced proliferation and interfered with signaling pathways to suppress steroidogenic activity. Overall, the data showed that the neonatal period is a sensitive window of exposure to isoflavones and support the view that both genistein and daidzein are responsible for biological effects associated with soy-based diets.