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Keratinocytes respond to skin irritation and injury by cytokine release and a rapid but transient activation of arachidonic acid metabolism along both the cyclooxygenase and lipoxygenase pathways. In the first part of this article results are reviewed indicating that the release of pro-inflammatory mediators such as eicosanoids and interleukin-1 from keratinocytes provides a suitable in vitro parameter of irritancy. Based on this response an assay system has been established which may partially replace animal tests such as the Draize test. A permanent overactivation of arachidonic acid metabolism appears to be a driving force of tumor development in both experimental animals and man. Inhibition of the enzymes involved (such as cyclooxygenases by nonsteroidal antiinflammatory drugs) provides, therefore, a powerful and promising measure of cancer chemoprevention. The state of the art in this rapidly developing field is briefly reviewed in the second part of this article.

Using a combination of conventional screening procedures and polymerase chain reaction cloning, we have isolated a cDNA encoding an epidermis-type 12-lipoxygenase (e12-lipoxygenase) from mouse epidermis. The open reading frame corresponds to a protein of 662 amino acids and was found to be 99.8% identical to the ORF of an epidermal lipoxygenase gene Aloxe, described recently [Van Dijk et al. (1995) Biochim. Biophys. Acta 1259, 4-8]. When expressed in human embryonic kidney cells the recombinant protein could be shown to synthesize 12(S)-HETE from arachidonic acid. By fluorescence in situ hybridization the e12-lipoxygenase gene was localized to chromosome band 11 B1-B3.

In mouse skin, tumor development promoted by 'non-genotoxic' carcinogens is closely related to the wound response. In both cases endogenous factors such as cytokines and eicosanoids released primarily from 'activated keratinocytes' play a key role as mediators of inflammation and cellular hyperproliferation. The liberation of interleukin-1 alpha and arachidonic acid from human keratinocytes has been used as an in vitro parameter of irritancy. The results (from experiments with 15 different chemicals) being validated at present in a clinical study indicate a quantitative relationship between irritancy in vivo and mediator release in vitro. In the course of experimental skin carcinogenesis an overproduction of eicosanoids due to a constitutive overexpression of the corresponding enzymes (i.e. PGH synthase-II and 8- and 12-lipoxygenase) is observed. Enzyme inhibitors, for instance nonsteroidal antiinflammatory drugs (NSAIDs), exert a strong tumoristatic effect. Thus, the approach of multistage skin carcinogenesis provides a suitable animal model for a mechanistic evaluation and further improvement of chemopreventive measures such as the inhibition of colorectal tumor development in humans by NSAIDs ('aspirin effect').

An anti-tumor-promoting effect of indomethacin and related nonsteroidal anti-inflammatory drugs (NSAIDs) as well as the ability of the tumor promoter 12-O-tetradecanoylphorbol-13- acetate (TPA) to increase the level of prostaglandins in murine keratinocytes and mouse epidermis in vivo has been repeatedly documented. Here, the expression of prostaglandin H synthase (PGHS) isozymes, which are major targets of NSAIDs, was investigated in different stages of tumor development in mouse skin. Mouse epidermis in vivo constitutively expressed PGHS-1. PGHS-1 steady-state levels remained unchanged upon induction of acute or chronic epidermal hyperplasia by TPA and in papillomas and carcinomas generated by the initiation-promotion procedure, with 7,12-dimethylbenz[a]anthracene as initiator and TPA as promoter. Thus, the elevated prostaglandin level in the acute hyperplastic epidermis was very likely due to PGHS-2 induction. Repeated applications of TPA resulted in stationary hyperplasia and downregulation of PGHS-2 expression and prostaglandin levels, suggesting that the epidermis had adapted to the TPA stimulus. In papillomas and carcinomas, however, constitutive overexpression of PGHS-2 was found, with a large amount of prostaglandin E2 and prostaglandin F2 alpha. Keratinocyte cell lines corresponding to different stages of tumor development also constitutively over-expressed PGHS-2. Considered with inhibitor studies, these data suggest that PGHS-2 has a critical role in skin carcinogenesis. The anti-tumor-promoting effect of the PGHS inhibitor indomethacin is specifically reversed by prostaglandin F2 alpha, indicating that this prostaglandin type has a significant role in tumor development.

This study is aimed at the development of a cell culture assay which may supplement or replace the animal Draize skin irritancy test. Using human keratinocytes, the measurement of proinflammatory eicosanoid and interleukin-1 alpha release and of the impairment of cell viability have provided a suitable in vitro/in vivo correlation for at least three surfactants. The in vitro study has been extended using structurally unrelated, pharmacologically relevant compounds including ethanol, glycerol, cyclohexanol, acetone, benzoic acid, phenol, acrylamide, triethanolamine, Tween 80, sodium dodecyl sulfate, benzalkonium chloride, NiSO4, SnCl2, and ZnCl2. Time- and dose-response studies were used to establish half-maximal stimulatory (SC50) and inhibitory (IC50) as well as 10-fold stimulatory (ED10) concentrations for arachidonic acid release, cytotoxicity, and IL-1 alpha release, respectively. Based upon these values a similar ranking has been obtained for the mildly acting acetone, ethanol, glycerol, and Tween 80 and for the severely acting A23187, benzalkonium chloride, and sodium dodecyl sulfate. With respect to all other test compounds, substantial variation occurred indicating that all three test parameters provide a more complete characterization of the test compound's potency than a single endpoint. These data are ready to be validated by a controlled clinical study aimed at a qualitative and quantitative evaluation of the symptoms of skin inflammation in volunteers.

The xanthate tricyclodecan-9-yl-xanthogenate (D609) displays antiviral and antitumoral properties that are inversely proportional in vitro to the serum concentration. Accordingly, it has been found that D609 binds to serum albumin. Recently, we have reported that D609, in combination with undecanoic acid, has a synergistic antiviral effect, which appears, as shown here, to be due to competition for the same binding domain on serum albumin. Furthermore, undecanoic acid fosters the binding of D609 to the cell. Both the competition of D609 with monocarbonic acid for binding on serum albumin and the enhanced binding of xanthate to the cell are dependent, in accordance with previously reported results, on the chain length of the fatty acids. Eleven to 14 C-atoms (undecanoic, lauric and myristic acid) were found to be appropriate while shorter (C6) and larger (C18) monocarbonic acids were shown to lack synergistic properties.

The growth of vesicular stomatitis virus (VSV) can be inhibited by the antiviral compound tricyclo-decane-9-yl-xanthogenate (D609). On analysing the antiviral mechanism we found no effect on the primary transcription of infecting VSV genomes. In contrast, the processes of replication and transcription during late stages of infection were inhibited. Despite the synthesis of all five virus-coded proteins (41% to 56% of the uninhibited control), as shown by labelling with [35S]methionine, the phosphorylation of the non-structural (NS) protein was reduced in the presence of the xanthate by a factor of at least 17. The pattern of phosphorylation of the bulk of cellular proteins remained unaltered under the same conditions. A relation between a possible loss of biological activity of the NS protein owing to the lack of phosphorylation and the decreased VSV RNA synthesis is suggested.

The binding of growth factors to the cellular receptors elicits the phosphorylation of proteins which transmit growth signals to the nucleus [E. Rozengurt (1986) Science 234, 161-166]. Both the tyrosine-specific kinase (growth factor receptor) and the threonine-serine phosphorylating protein kinase C (pkC) become activated upon binding of the epidermal growth factor (EGF) to its receptor. Here we describe the selective inhibition of the pkC activation by tricyclodecane-9-yl-xanthogenate (D609) in the presence of unsuppressed receptor tyrosine autophosphorylation. As a consequence the affinity of EGF to the receptor was not down-regulated and the complex failed to be internalized.

Xanthate compounds have been shown to exhibit antiviral activity against various DNA and RNA viruses under acidic pH conditions. It is now possible to utilize the unique broad range antiviral spectrum of these compounds under physiological pH conditions (pH 7.4) by simultaneous administration of certain ionic detergents. When used in conjunction with tricyclodecan-9-yl-xanthate (D609), sodium deoxycholate, sodium dodecylsulfate and certain fatty acids, which have no antiviral activity of their own, inhibit the replication of various DNA and RNA viruses (such as herpes simplex, vesicular stomatitis and Coxsackie B 4) in vitro at pH 7.4. Among saturated fatty acids of various chain lengths there was a marked size restriction in that the efficiency of undecanoic acid (11 C atoms) was three orders of magnitude greater than that of shorter (6 C atoms) or longer (18 C atoms) monocarbonic acids. Dose-response kinetics revealed a synergistic interaction between the xanthate and the monocarbonic acid. A dose that inhibited the replication of herpesvirus by a factor of 1000 still permitted mitotic activity in uninfected growing control cultures.