The neutral red cytotoxicity assay was used in vitro to evaluate the potencies of phenylpropanolamine (PPA), nicotine, caffeine, some of their metabolites, and related chemicals.

The human cell types used as targets included fibroblast (HFF), melanoma (SK-Mel/27), and hepatoma (HepG2) cell lines and early passage endothelial (ENDO) cells and keratinocytes (NHEK).

For all of these cells, nicotine was more cytotoxic than cotinine, its major metabolite.

In turn, cotinine was more cytotoxic than chemically related compounds such as nicotinic acid and nicotinamide.

Nicotine, but neither cotinine, nicotinic acid, nor nicotinamide, induced cytoplasmic vacuolization in all the cell types tested. Except for the ENDO cells, caffeine and its metabolite, theophylline, showed approximately equivalent cytotoxic potencies.

However, for the ENDO cells, caffeine was more cytotoxic than theophylline.

Furthermore, the ENDO cells were 2-3 times more sensitive to caffeine and theophylline than were the other cell types.

The phenylpropanolamine induced cytoplasmic vacuolization only in the ENDO cells. Combinations of caffeine + PPA interacted synergistically in their cytotoxicity towards the HepG2 cells; a similar synergistic interaction was not noted with the ENDO cells.

Fourteen cosmetic products reflecting a range of irritancy levels were evaluated for cutaneous irritation potential using dermal equivalent (DE) and skin equivalent (SE) kits. Our two in vitro models are presented in 12 culture inserts (Transwell, Costar) allowing air-liquid interphase culture and topical application.

Dermal equivalent includes a collagen-glycosaminoglycans-chitosan porous matrix populated by human normal fibroblasts and skin equivalent is realized by seeding keratinocytes onto the DE.

The pure, cosmetic products were applied in triplicate by topical application (10 ul) onto a surface delimited by a silicone assay ring placed onto DE and SE.

After 24 hr contact with the cosmetic products, the residual cellular viability was measured using a MTT test on treated and untreated tissues.

The purpose of this preliminary validation study was to evaluate to what extent the in vitro results can predict in vivo skin irritation.

Consequently, 11 cosmetic products with known Draize irritation classes were tested on DE.

Ten of 11 were correlated if we considered only the irritancy potential prediction (irritant or nonirritant) but considering the binary correlation with three classes of irritation (irritant, slightly irritant, or nonirritant) 8 of 11 were correlated.

Moreover, the parameters of validation were calculated. Second, three cosmetic products with known Draize primary irritation index (PDII) were tested both on DE and SE. The correlation of the in vitro MTT values to the in vivo data using a regression line was found to be r = 0.99 for DE and r = 0.99 for SE.

These preliminary results are encouraging and suggest that the two models, DE kit and SE kit, could be used as in vivo alternative methods after a complete validation study involving the testing of the different chemical classes and various cosmetic forms.

It is known that alkylating processes occur after exposure to sulfur mustard (HD). Although direct alkylation of DNA and RNA has been widely described, we hypothesize additional alkylation events of potential importance in skin injury.

We have determined by immunocytochemistry levels of interleukin-1 b (IL-1b ) in cultures of normal human epidermal keratinocytes (NHEK) after exposure to HD.

The expression of IL-1b in NHEK was found to be related to cell culture donor age. In neonatal NHEK exposed to HD (2 mM), IL-1b response is increased.

Electron paramagnetic resonance (EPR) spectroscopy was used to show the formation of EPR detectable, g=2.04, feature characteristic of iron-nitrosyl complex formation, and the generation of this induced complex by NHEK exposed to HD (1 mM for 18 hours) was blocked by Nw -Nitro-L-arginine (L-NOARG), a competitive inhibitor of nitric oxide synthase (NOS). Nitric oxide (·NO) has been implicated as the effector molecule that mediates IL-1b (Corbett et al, 1993).

Our results show the release of nitric oxide during cytokine expression, IL-1b , when keratinocytes ae exposed to HD.

The combination of the nitric oxide with the chloride (Cl- ) released from sulfur mustard ([ClCH₂CH₂]₂S) on cyclization to the sulfonium ion may lead to the formation of nitrosyl chloride (NOCl), a known potent alkylating agent.

If NOCl is formed as a result of HD exposure, then it may play a role in the skin injury.