SVK14 Cells Research Articles

Molecular Genetic Dissection of Inflammatory Linear Verrucous Epidermal Naevus Leads to Successful Targeted Therapy

Molecular Genetic Dissection of Inflammatory Linear Verrucous Epidermal Naevus Leads to Successful Targeted Therapy

The effect of silver nanoparticles and silver ions on mammalian and plant cells in vitro

Silver nanoparticles (AgNPs) are the most frequently applied nanomaterials. In our experiments, we tested AgNPs (size 27 nm) manufactured by the Tollens process. Physico-chemical methods (TEM, DLS, AFM and spectrophotometry) were used for characterization and imaging of AgNPs. The effects of AgNPs and Ag+ were studied in two experimental models (plant and mammalian cells). Human keratinocytes (SVK14) and mouse fibroblasts (NIH3T3) cell lines were selected to evaluate the cytotoxicity and genotoxicity effect on mammalian cells. Higher sensitivity to AgNPs and Ag+ was observed in NIH3T3 than in SVK14 cells. AgNPs accumulated in the nucleus of NIH3T3 cells, caused DNA damage and increased the number of apoptotic and necrotic cells. Three genotypes of Solanum spp. (S. lycopersicum cv. Amateur, S. chmielewskii, S. habrochaites) were selected to test the toxicity of AgNPs and Ag+ on the plant cells. The highest values of peroxidase activity and lipid peroxidation were recorded after the treatment of S. habrochaites genotype with AgNPs. Increased ROS levels were likely the reason for observed damaged membranes in S. habrochaites. We found that the cytotoxic and genotoxic effects of AgNPs depend not only on the characteristics of nanoparticles, but also on the type of cells that are treated with AgNPs.

Reprint of: Cytotoxicity, cell uptake and microscopic analysis of titanium dioxide and silver nanoparticles in vitro

Commercially manufactured nanomaterials are used massively for modification of products of everyday use, including products intended for children. Therefore their potential risks have to be ultimately studied. Aside from toxicity of nanomaterials with known specific parameters, the end-consumer is potentially endangered by materials with unknown specification. Commercially available products are not usually accompanied by parameter/specification sheet providing the consumer with sufficient chemico-physical parameters allowing the evaluation of possible toxic effects.The aim of this work was to evaluate the declared parameters of commercially available TiO2 and Ag NPs employing chemico-physical methods and consequently in vitro cytotoxicity and genotoxicity tests performed on non-cancer cell lines. Based on the results of our complex study we can conclude that the data provided by the producers are not in good agreement with the performed measurements. Furthermore, all tested NPs penetrated into the SVK14 cells and all NPs had significant effect on the kinetics of ROS production in all cell lines (note: the ROS production has not been established as the major mechanism of cell damage elicited by Ag NPs). The study revealed greater cytotoxic potential of Ag NPs in comparison with TiO2 NPs and all of the studied NPs caused significant DNA damage.

Cytotoxicity, cell uptake and microscopic analysis of titanium dioxide and silver nanoparticles in vitro

Commercially manufactured nanomaterials are used massively for modification of products of everyday use, including products intended for children. Therefore their potential risks have to be ultimately studied. Aside from toxicity of nanomaterials with known specific parameters, the end-consumer is potentially endangered by materials with unknown specification. Commercially available products are not usually accompanied by parameter/specification sheet providing the consumer with sufficient chemico-physical parameters allowing the evaluation of possible toxic effects.The aim of this work was to evaluate the declared parameters of commercially available TiO2 and Ag NPs employing chemico-physical methods and consequently in vitro cytotoxicity and genotoxicity tests performed on non-cancer cell lines. Based on the results of our complex study we can conclude that the data provided by the producers are not in good agreement with the performed measurements. Furthermore, all tested NPs penetrated into the SVK14 cells and all NPs had significant effect on the kinetics of ROS production in all cell lines (note: the ROS production has not been established as the major mechanism of cell damage elicited by Ag NPs). The study revealed greater cytotoxic potential of Ag NPs in comparison with TiO2 NPs and all of the studied NPs caused significant DNA damage.

The chemical chaperone trimethylamine N-oxide ameliorates the effects of mutant keratins in cultured cells

Conflicts of interest: none declared. Sir, Mutations in keratin genes are the cause of at least 19 distinct disorders, many of which affect the skin and its appendages.1 The clinical symptoms vary according to the expression pattern of the mutated keratin but typically involve cell fragility and hyperkeratosis. Although keratin disorders are rarely life threatening, they can be painful and debilitating and most treatments are limited to pain relief and prevention of infection. The observation that many keratin mutations are associated with cytoplasmic keratin aggregates led us to hypothesize that keratin disorders are protein‐folding disorders and that chemical chaperones, which promote normal protein folding, will reduce the cellular effects of the mutant keratins.2 To test this hypothesis we examined the ability of the chemical chaperone trimethylamine N‐oxide (TMAO) to reduce the effects of hyperosmotic stress in cells expressing mutant keratins. We first examined the effect of hyperosmotic stress on a cell line derived from a patient with epidermolysis bullosa simplex (KEB‐1; K5:Glu475Gly).3 Incubation of KEB‐1 cells with medium containing 300 mmol L−1 sorbitol for 30 min led to collapse of the keratin cytoskeleton and the formation of cytoplasmic keratin aggregates in more than 20% of the cells. In contrast, keratin aggregates formed fewer than 5% of controls cells (SVK14 cell line) following similar treatment. Pretreatment of KEB‐1 cells with TMAO reduced the number of cells with keratin aggregates to control levels (Fig. 1). A decrease in the number of keratin aggregate‐containing cells was noted after prolonged exposure (> 30 min) of KEB‐1 cells to hyperosmotic conditions. This could indicate that cells containing aggregates were lost from the culture dish or that the keratinocytes were re‐establishing their keratin cytoskeleton. To distinguish between these possibilities, KEB‐1 and SVK14 cells were incubated with sorbitol for various times and the number of cells remaining attached to the plate determined. There was a time‐dependent decrease in the number of KEB‐1 cells indicating that cells containing keratin aggregates were preferentially lost from the culture dish. No effect on cell number was observed in control cells. Pretreatment with TMAO protected KEB‐1 cells from hyperosmotic‐induced cell loss (Fig. 1).

Intronic UGG repeats coordinate splicing of CD44 alternative exons v8 and v9

Alternative CD44 exons v8, v9, and v10 are spliced as a block in epithelial cells (for example SVK14 cells), but can be skipped as a block by other cells. Using a minigene approach, we show that downstream intronic UGG repeats participate in activation of v8 exon splicing in SVK14 cells. The repeats can activate splicing of a heterologous exon in SVK14 cells and act additively with a previously described v8 exon splicing enhancer in this context. An alternative v9 exon 5′ splice site used by some cells to make an aberrant transcript is repressed by an immediately downstream (UGG) 3 sequence in SVK14 cells. We conclude that UGG repeats both activate v8 exon splicing and repress use of the alternative v9 exon 5′ splice site in SVK14 cells, thus participating in the coordination of correct epithelial cell splicing of the v8–10 block.

Effect of sulphur mustard on human skin cell lines with differential agent sensitivity

The ability of sulphur mustard (HD) to induce DNA damage places limits on the efficacy of approaches aimed at protecting human cells from the cytotoxic effects of HD using a variety of protective agents such as thiol-containing esters and protease inhibitors. In the present study, potential alternative strategies were investigated by examining the differential effects of HD on G361, SVK14, HaCaT and NCTC 2544 human skin cells. The G361 cell line was more resistant to the cytotoxic effects of HD than the NCTC, HaCaT and SVK14 cell lines at HD doses of >3 and <100 microM HD as determined by the MTT assay. At 72 h after exposure to 60 microM HD there was up to an 8.8-fold difference (P < 0.0001) between G361 and SVK14 cell culture viability. Buthionine sulphoximine (BSO) pretreatment increased the sensitivity of all four cell lines to HD. A substantial proportion of the resistance of G361 cells to HD was attributable to BSO-mediated effects on antioxidant-mediated metabolism, although G361 cultures still retained a high degree of viability at 30 microM HD following BSO pretreatment. Cell cycle analysis confirmed that SVK14 cells were relatively more sensitive to HD, as shown by the 2.1-fold reduction (P < 0.0001) in the percentage of cells in G0/G1 phase 24 h after HD exposure compared with control cultures. This compared well with a 1.2-fold increase (P < 0.05) in the percentage of G361 cells in G0/G1 phase following HD exposure, suggesting the existence of a more efficient G0/G1 checkpoint control mechanism in this cell line. Manipulation of the cell cycle using various modulating agents did not increase the resistance of cell lines to the cytotoxic effects of HD.

SVK14 cells express an MCH binding site different from the MCH 1 or MCH 2 receptor

Melanin-concentrating hormone (MCH) is a cyclic peptide, mainly involved in the regulation of skin pigmentation in teleosts and feeding behavior in mammals. The human keratinocyte SVK14 cell line has been previously shown to express binding sites for the MCH analog [ 125 I] -[ Phe 13,3 -iodo-Tyr 19] MCH. We report here that: (1) this binding site similarly recognized [ 125 I] -[3 -iodo-Tyr 13] MCH; (2) its pharmacological profile clearly differed from those observed at the two human MCH receptor subtypes, MCH 1-R and MCH 2-R; (3) MCH did not induce any effect on second messenger systems (including cAMP, calcium, and MAP kinase signaling pathways), and (4) no mRNAs corresponding to the MCH receptors were found. In conclusion, the binding site characterized in the SVK14 cell line is distinct from the MCH 1 and MCH 2 receptors and deserves therefore further investigation.

Characterization of two monoclonal antibodies against the RON tyrosine kinase receptor.

RON is a receptor protein tyrosine kinase belonging to the hepatocyte growth factor (HGF) receptor family. Using Récepteur d'Origine Nantais (RON) transfected cell lines, Macrophage Stimulating Protein (MSP) was identified as the ligand of RON. RON is synthesized as a single chain precursor, which subsequently is cleaved to yield a disulfide-linked heterodimer, with a 40-kDa alpha chain and a 150-kDa beta chain. Activation of RON by MSP results in cell migration, shape change, and proliferation. The present work centers on the production and characterization of two monoclonal antibodies (MAbs) to RON called ID-1 and ID-2. Antibodies were generated by immunization of mice with Madin-Darby Canine Kidney (MDCK) cells expressing human RON (clone RE7). Both antibodies recognized the mature and precursor form of RON. The specificity of the anti-RON antibodies was confirmed using a hepatocarcinoma cell line HepG2 expressing both task MET and RON receptors. Specific immunoprecipitation with ID-1 and ID-2 or anti-MET antibody followed by Western blotting under reducing conditions with rabbit polyclonal antibodies against RON and MET showed that our anti-RON antibodies recognize specifically the RON receptor. Ligand binding experiments showed that both antibodies are able to block the binding of radiolabeled MSP to RON and showed also that the antibodies recognize two different epitopes in the molecule. The blocking of MSP binding to RON by the anti-RON antibodies was confirmed by inhibition of cell migration induced by MSP in HT-29-D4 cells. Significant immunostaining was not observed in any subpopulation of whole blood with either ID-1 or ID-2. We analyzed the expression of RON receptor in a number of human hematopoietic and nonhematopoietic cells lines by flow cytometry. We found a strong mean of fluorescence intensity (MFI) in colon adenocarcinoma cells SW620 and HT-29-D4, low MFI in SVK14 and HepG2 cells, and no immunostaining in melanoma, lymphoma, and leukemia cells. Immunohistochemistry revealed that RON was expressed in germinal centers of tonsil, in skin, small intestine, and colon. These antibodies defined RON as CDw136 during the last leucocyte typing VI.

Melanin-Concentrating Hormone Binding Sites in Human SVK14 Keratinocytes

Melanin concentrating hormone (MCH) is a cyclic peptide which regulates a broad array of functions in the mammalian brain and it may act as a paracrine factor in peripheral organs. In these studies a radiolabeled MCH derivative, the [125I]-[Phe13, Tyr19]-MCH, was synthesized and used as a tracer to perform binding experiments. A number of human or rodent cell lines displayed specific binding with [125I]-[Phe13, Tyr19]-MCH, the highest binding capacity being observed with human SVK14 keratinocytes. Saturation binding analysis with SVK14 cells indicated about 10,000 MCH binding sites per cell and a Kd of 0.7 nM for [125I]-[Phe13, Tyr19]-MCH. Surprisingly, the iodinated [Phe13, Tyr19]-MCH displayed about 10-fold higher affinity (Ki ∼ 3.0 nM) for the putative MCH receptor than the noniodinated form (Ki ∼ 25-30 nM). Competition binding analyses comparing various MCH-related peptides revealed a similar low binding potency for all these peptides (Ki ∼ 65-160 nM). Strikingly, rat ANP and rat/human CNP but not rat BNP displaced [125I]-[Phe13, Tyr15]-MCH with Ki ∼ 210-365 nM and may be due to topological similarities instead of partial sequence identities between MCH and some of the natriuretic peptides. However, other peptides such as CRF, αMSH, Arg-vasopressin, and MGOP-peptide I did not compete with the radioligand. Finally, the molecular mass of the MCH binding sites on SVK14 cells was estimated to be 47 kDa by crosslinking and SDS-PAGE experiments. Taken together, our data revealed the widespread expression of MCH binding sites on mammalian cells, particularly on skin carcinoma cells. However, the low affinity of these sites for the native MCH and MCH-related peptides as well as competitivity with ANP and CNP indicates that further biochemical and functional characterizations are needed to validate them as genuine physiological MCH receptors.

Evaluation in vitro of epidermal cell keratinization

Rhodamine B has been used as a histopathological stain for keratinization and cornification. Its ability as an in vitro indicator of the degree of epidermal keratinization was investigated in these preliminary studies. An immortalized human keratinocyte cell line, SVK-14, was evaluated as an alternative to primary human keratinocytes. The influence of extracellular calcium levels was evaluated alongside the effects of exposure to 1,25 (OH) 2 vitamin D 3 in serum-free and serum-containing media. Alamar blue (AB) conversion was used to measure changes in cellular reductive potential, and the amount of bound Rhodamine B relative to total protein per well was taken as an indicator of keratinization. Exposure to 1,25 (OH) 2 vitamin D 3 for 7 or 10 days did not increase Rhodamine B binding to confluent SVK-14 cultures, regardless of calcium concentration. Variation in Rhodamine B dye-binding to cells made it difficult to interpret the data. In addition, concern regarding the ability of SVK-14 cells to differentiate suggests that further studies need to be performed using normal human keratinocytes to validate this in vitro endpoint, with epidermal growth factor, insulin and hydrocortisone removed from the media to enhance epidermal differentiation.

E-Selectin Binds to Squamous Cell Carcinoma and Keratinocyte Cell Lines

E-selectin is an endothelial adhesion molecule that binds carbohydrate epitopes on leukocytes and has been implicated in a potential pathway of tumor metastasis. Keratinocyte cell lines express similar carbohydrate epitopes, one of which, sialyl Lewis X (SL-X) is a ligand for E-selectin and is also expressed by squamous cell carcinomas (SCC) in situ. The functional role of keratinocyte selectin ligands was investigated using a soluble E-selectin chimaeric protein (pE-sel-Ig) containing pig lectin-like and epidermal growth factor-like domains fused to human IgG. After incubation of keratinocyte cell lines (A431 and SVK14) and normal keratinocytes with pE-sel Ig, binding was quantified by flow cytometry. Frozen sections of SCC were overlaid with pE-sel Ig and binding was visualized immunoenzymatically. Immunolabeling was undertaken using monoclonal antibodies (CSLEX-1 and HECA-452), which label E-selectin ligands including sialyl Lewis X. E-selectin bound strongly to A431 and SVK14 cells; the degree of binding paralleled staining intensity with CSLEX-1 antibody. HECA-452 antibody stained A431 cells strongly but SVK14 cells only weakly. Normal keratinocytes and normal epidermis did not express CSLEX-1 or HECA-452 antigens or bind E-selectin. Serial sections of SCC revealed close correlation between fusion protein binding and antibody staining. Antibody pretreatment of tumor sections with CSLEX-1 blocked fusion protein binding, whereas HECA-452 antibody only slightly reduced fusion protein binding. pE-sel Ig pretreated with YT11.1 antibody failed to bind to A431 or SVK14 cells or to SCC. These studies provide functional evidence that SL-X/E-selectin pathways may be important in SCC metastasis and that A431 and SVK14 cells provide a good model to investigate these mechanisms.

Fumaric acid derivatives evoke a transient increase in intracellular free calcium concentration and inhibit the proliferation of human keratinocytes

Systemic administration of fumaric acid (FA) derivatives was originally an empirical antipsoriatic treatment, which showed promising clinical results. In the present study, FURA-2-loaded suspensions of cultured normal keratinocytes and SV40-transformed keratinocytes (SVK-14 cells) were used to study the effects of FA derivatives on the intracellular free calcium concentration ([Ca2+]i). Monomethylfumarate (MMF), dimethylfumarate (DMF) and monoethylfumarate (MEF) induced a rapid, transient [Ca2+]i increase in both cell types. This immediate increase reached maximal values of 396 nmol/l 10s after addition of MMF, and fell to basal values within 90-120 s (173 nmol/l for normal keratinocytes and 68 nmol/l for transformed keratinocytes). This increase was not affected by the prior addition of EGTA, indicating that FA derivatives released Ca2+ mainly from intracellular stores into the cytoplasm. Subsequently, dose-dependent inhibitory effects of FA derivatives on keratinocyte proliferation were demonstrated. The results of these experiments revealed that DMF was the most potent, MMF and MEF intermediate, and FA and malonic acid the least potent growth inhibitors. These antiproliferative effects of FA derivatives might be linked to the observed, transient [Ca2+]i elevations.

Plasminogen activators are involved in keratinocyte and fibroblast migration in wounded cultures in vitro

During skin repair both keratinocytes and fibroblasts migrate into the wounded area. In the process of cell migration, controlled proteolytic degradation of the extracellular matrix occurs. It has been suggested that the plasminogen activator system is involved in such proteolytic processes occurring during wound healing. The role of plasmin, urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA) in keratinocyte and fibroblast migration in vitro was examined in the present study. Confluent cultures of normal human keratinocytes, human squamous cell carcinoma (SCC-4), SV40 transformed keratinocytes (SVK-14) and human fibroblasts were mechanically wounded and the repopulation of the denuded area was examined. Migration of cells into the denuded area could be inhibited by neutralizing antibodies against t-PA or u-PA, or the serine protease inhibitor Trasylol in all cell types studied. For detailed study on involvement of u-PA and t-PA in migration processes wounded cultures of SVK14 cells were used. Both t-PA and u-PA activity could be detected at the migrating edge of SVK14 cultures as revealed by zymography and by immunocytochemistry using polyclonal antibodies. Our results demonstrate the direct involvement of not only u-PA but also t-PA in migration of keratinocytes and fibroblasts in wounded cultures in vitro.

Use of human skin cell cultures for the estimation of potential skin irritants

We have investigated the use of cell cultures derived from human skin (keratinocytes and fibroblasts) and of cultured SV40 transformed human keratinocytes (SVK14 cells) for in vitro screening for skin toxicity. To assess the cytotoxic effects of a number of surfactants the following cytotoxicity assays were performed: (1) changes in cell morphology and cell number in confluent cultures; (2) inhibition of cell proliferation; (3) modulation of interleukin-6 (IL-6) production. The data obtained in vitro were compared with the results of an in vivo study (Tupker et al. Contact Dermatitis 1989, 20, 108–114), in which visual scoring and measurement of transepidermal water loss were taken as measures of skin irritancy. The results of the morphological and proliferation studies in vitro indicated a similar ranking order in skin irritancy as that found in the in vivo study. Furthermore, IL-6 production by cultured skin cells was increased on administration of nearly all of the surfactants tested. For a number of surfactants the increase in IL-6 production occurred at much lower concentration than those necessary to induce cytotoxic effect, suggesting that monitoring the IL-6 production on administration of skin irritating agents could be used as a very sensitive test.

Induction of terminal differentiation in cultured human keratinocytes by polychlorinated aromatic hydrocarbons as measured by cell size analysis

Polychlorinated aromatic hydrocarbons modulate the proliferation and differentiation of human epidermal cells in vivo and in culture. One of the earliest events in the process of terminal differentiation is the increase in cell size. In this report the usefulness of morphometric cell size analysis as a quantifiable marker for chemical-induced differentiation was examined. Concentration-related increases in cell size distribution were induced by 2,3,7,8-tetrachlorodibenzo- p-dioxin (2,3,7,8-TCDD) and 2,3,4,7,8-pentachlorodibenzofuran in normal human keratinocytes and cells from an SV40-transformed keratinocyte cell line (SVK14) whereas the analog 1,2,3,4-tetrachlorodibenzo- p-dioxin did not affect the cell size distribution up to a concentration of 100 n m. The minimal effective concentrations of five 2,3,7,8-substituted polychlorinated dibenzo- p-dioxins/dibenzofurans and a coplanar polychlorbiphenyl necessary to induce an increase in cell size distribution were determined in SVK14 cells. It was found that the potency of these compounds relative to that of 2,3,7,8-TCDD correlated well with the toxicity equivalency factors observed in other test systems. This indicates that the keratinocyte cell assay is a useful method for establishing the relative potency of various “dioxins” and their mixtures.

Interferon-Gamma Activates Co-Ordinate Transcription of HLA-DR, DQ, and DP Genes in Cultured Keratinocytes and Requires De Novo Protein Synthesis

The purpose of this study was to determine the effect of interferon-gamma on keratinocyte major histocompatibility complex class II gene transcription. Transformed human foreskin keratinocytes (SVK14 cells) were incubated with recombinant IFN-gamma in the presence or absence of the protein synthesis inhibitor cycloheximide. Total cellular RNA was extracted from the cells and Northern blot analysis carried out using cDNA probes for all the functional class II genes. We report that 1) there is co-ordinate activation of all the class-II genes; 2) the rate of transcription varies between gene loci after activation; and 3) de novo protein synthesis is required for IFN-gamma activation of class II transcription.

Transglutaminases in Normal and Transformed Human Keratinocytes in Culture

The transglutaminases of cultured normal and transformed human keratinocytes (line SV-K14) are characterized. Both cell types display two forms of the enzyme, one of which is cytosoluble (TGc) and the other which is associated with the plasma membrane (TGm). Normal keratinocytes contain predominantly TGm, and SV-K14 cells mainly TGc. The ratio of TGm to TGc can be modulated by the culture conditions and correlates with the competence of the cells to form a cornified envelope. TGm and TGc differ in their biochemical and immunological properties. SDS electrophoresis reveals apparent molecular weights of 92 and 85 kD, respectively. Only the activity of TGc is inhibited in the presence of guanosine 5'-triphosphate. Their response to Ca2+ is different: TGc exhibits a sigmoidal activation kinetics with an A50 value of about 200 microM, whereas the kinetics for TGm is hyperbolic with an A50 value of 75 microM. TGm reacts with a monoclonal antibody raised against epidermal "particulate" transglutaminase, and TGc with a polyclonal antibody raised against guinea pig liver transglutaminase. These reactions are very specific and no cross-reaction occurs. The coappearance of TGm with a proteolytic fragment (Mr 82,000) in the cytosol and intracellular particulate fraction of normal human keratinocytes is probably a preparation artifact.

Lateral mobility of plasma membrane lipids in normal and transformed keratinocytes

In this study we have examined possible differentiation-dependent modulations in plasma membrane lipid properties in normal keratinocytes, SV-40 transformed keratinocytes (SVK14) and a number of squamous carcinoma (SCC) cells. In normal keratinocytes the lateral diffusion coefficient of plasma membrane lipids (D) differs significantly for cells cultured permanently under low and normal Ca2+-conditions (5.16 x 10(-9) and 3.27 x 10(-9) cm2/s, respectively). When differentiation is induced by exposing low Ca2+-cultured cells to normal Ca2+ concentrations D increases to 7.07 x 10(-9) cm2/s during the initial hours of differentiation followed by a gradual sustained decrease to values also observed in cells cultured permanently under normal Ca2+-conditions. In SCC and SVK14 cells a similar initial transient increase in lateral lipid mobility is observed upon initiation of differentiation, but, in contrast to normal keratinocytes, no sustained decrease in D is seen upon prolonged culturing under normal Ca2+ conditions. The results indicate that the deficiency of the transformed cells to respond to Ca2+-induced differentiation might involve transformation-dependent alterations in membrane structure and function.

(2) Comparative toxicity of antimicrobial agents on transformed human keratinocytes

Topical antibiotics and antiseptics are widely used to prevent and treat bacterial infections in wounds and ulcers. Experimental data suggest that some antimicrobial agents may have an adverse effect on several aspects of the tissue repair process, including retardation of wound epithelialization. The aim of this study was to investigate the comparative cytotoxic effects of a range of antiseptics and antibiotics using human keratinocytes transformed by Simian virus 40 (SVK14 cells). SVK14 cells were grown to semi-confluence by adding 6 × 1014 cells per petri dish. After 48 h the cells were exposed to serial dilutions of the therapeutic concentrations (shown in parentheses) of each of the following agents; hydrogen peroxide (3%), cetrimide (1%), sodium hypochlorite (o·5%), povidone iodine (4%), neomycin (1%), bacitracin (50 units/ml), polymyxin B sulphate (10000 units/ml). The cells were exposed to the drug for 15 min and then washed and incubated in the culture medium (RPMI 1640 plus 10% foetal calf serum) for 24 h. Dead cells were then washed off and the viable adherent cells were trypsinized and counted in a Coulter counter. At therapeutic concentrations none of the antibiotics was found to be cytotoxic, whereas all of the antiseptics produced 100% killing of SVK14 cells. Dilutions of therapeutic concentrations of the antiseptics ranging between 1000- and 20000-fold (depending on the agent) were needed in order to achieve no-effect levels. Calculations based on 100% killing values for the antiseptics indicate that their order of toxicity from highest to lowest is: sodium hypochlorite, cetrimide, povidone iodine and hydrogen peroxide. Whilst it is appreciated that more work is needed in order to assess the exact relevance of these findings to clinical practice, it should be noted that SVK14 cells share many of the characteristics of normal keratinocytes and that these preliminary results are broadly in line with in vitro, in vivo and human volunteer findings made by other investigators studing antimicrobial toxicity profiles. We conclude that this cell line may be useful in studying the epithelial cytotoxicity of drugs in vitro and that care should be exercised in the selection of antimicrobial agents for use in wound management.