D.J. Benford

DEVELOPMENT OF AN OPTIMAL METHOD FOR THE CRYOPRESERVATION OF HEPATOCYTES AND THEIR SUBSEQUENT MONOLAYER CULTURE

Adult rat hepatocytes, of good viability, were routinely isolated using a versatile biopsy perfusion technique. In order to develop an optimal cryopreservation regimen we investigated a variety of factors including the cooling rate, the constituents of the medium, the cryoprotective agents and the thawing conditions. The preferred freezing medium consisted of Leibovitz L15 culture medium supplemented with 10% (v/v) foetal calf serum, 10% (v/v) tryptose phosphate broth, 20% (v/v) dimethylsulphoxide, 100 mug/ml streptomycin and 100 U/ml penicillin. A freezing rate faster than the generally used 1-2 degrees C/min appeared to be optimal for subsequent hepatocyte attachment and survival in culture. Handling conditions after thawing were important for maximal recovery of cells in culture. To assess the functional integrity of hepatocytes in monolayer culture after cryogenic storage a number of parameters were monitored. Biochemical assays included lactate dehydrogenase leakage, protein synthesis, cellular ATP and ADP content, glutathione (reduced form), cytochrome P-450 content, ethoxycoumarin-O-deethylase activity, glucuronide and sulphate conjugation capacity. Cytochrome P-450 content was maintained at levels comparable with those in freshly prepared cultures for up to 24 hr in culture; ethoxycoumarin-O-deethylase activity was also similar to that of freshly prepared cultures. Changes in various biochemical parameters indicated that cryopreservation resulted in subtle damage to hepatocytes and that even cells that excluded dye did not survive as well as non-frozen cells. When human hepatocytes were subjected to the method developed for rat hepatocytes, they survived the trauma of cryopreservation but fewer cells subsequently attached and spread out to form a monolayer culture. These results indicate that human hepatocytes are more sensitive to freezing under the conditions developed for rat hepatocytes. This may reflect a greater sensitivity of the cells to cryopreservation and/or that the cryogenic conditions were not optimal for human hepatocytes.

Use of human and rat keratinocyte cultures to assess skin irritation potential.

An in vitro cell culture approach was evaluated for its ability to provide data pertinent to the assessment of skin irritation potential. The hypothesis of this approach is that a direct toxic insult to the epidermal keratinocyte in vivo may lead to release of inflammatory mediators, which are responsible for initiation of a local primary skin irritant reaction. This paper presents data on the cytotoxic potential of a number of structurally unrelated chemicals (chloroform, 2-methoxyethanol, 2-butoxyethylacetate, toluene, 1-butanol, acetaldehyde, n-hexane, sodium dodecyl sulfate, benzalkonium chloride, silver nitrate, dibutyltin dichloride and tributyltin chloride). Cytotoxicity (neutral red uptake and intracellular acid phosphatase activity) of a number of structurally unrelated chemicals, representative of a wide range of skin irritation potential, was evaluated in cultures of rat and human epidermal keratinocytes. The sensitivity of human and rat keratinocytes to the test chemicals was very similar, irrespective of the endpoint of cytotoxicity. The neutral red uptake assay appeared more generally applicable to the diverse range of chemical structures represented in this study, since not all test chemicals elicited an early increase in intracellular acid phosphatase activity. The results were very encouraging, as a good correlation was evident between cytotoxicity in rat keratinocytes and the degree of erythema and oedema associated with an in vivo skin irritant response in rabbits. Keratinocyte cytotoxicity data may provide an indication of the potential of a chemical to induce a severe skin irritant reaction, or if a chemical is more likely to be a marginal or non-irritant. However, the data illustrate that such assays appear unable to discriminate correctly between more subtle classes of irritancy, such as non-irritant, mild, moderate or severe. Available human in vivo skin irritation data were insufficient to conclude which cell type is preferable for evaluation of human skin irritation potential.

Skin irritant-induced cytotoxicity and prostaglandin E2 release in human skin keratinocyte cultures

Damage to the skin induced by chemical irritants is associated with the release of arachidonic acid metabolites, such as prostaglandin E(2) (PGE(2)) which plays an important role in epidermal inflammation. This study investigated cytotoxicity and release of PGE(2) in human epidermal keratinocytes following an 18 hr exposure of confluent cultures to various skin irritants. The concentration-dependent release of PGE, into the extracellular medium appeared to fall into two categories, which was reflective of possible mechanisms of action. Potent skin irritants, such as phorbol-12-myristate-13-acetate, benzalkonium chloride and tributyltin chloride, elicited an increase in extracellular PGE(2) levels at concentrations that did not produce overt cell damage (uptake of neutral red at these concentrations was comparable to control levels). Non-irritants (2-methoxyethanol and 2-butoxyethyl acetate) and two less severe irritants (sodium dodecyl sulfate and acetic acid) stimulated release of PGE(2) only at concentrations that compromised cellular integrity (uptake of neutral red was at least 50% lower than that of control cultures).

Release of inflammatory mediators in human keratinocyte cultures following exposure to a skin irritant

The most common pathological response of the skin, associated with chemical-induced primary irritation, is inflammation. Release of inflammatory mediators occurs in primary irritant dermatitis in both laboratory animals and humans. Inflammatory mediators (prostaglandin E(2), leukotrienes C(4), D(4), E(4) and 15-hydroxyeicosatetranoic acid) were determined by radioimmunoassay, and leukotriene B(4) and the cytokine, interleukin 1alpha, were measured by enzyme-linked immunosorbent assay, in cultures of normal adult human keratinocytes. Release of inflammatory mediators into the culture medium was assessed at various time points following treatment of the cultures with the anionic surfactant sodium dodecyl sulphate (SDS), a known human skin irritant. A cytotoxic response was confirmed by the neutral red uptake assay after a 24-hr exposure to SDS. A dose-related release of inflammatory mediators was observed. The magnitude of the response varied between different mediators and as a function of time. The results show that release of inflammatory mediators occurs in human epidermal keratinocyte cultures, following chemical insult. Large inter-experimental variations in release of the various mediators probably preclude their use in a routine assay to determine irritation potential. In the case of SDS, release of inflammatory mediators is not a sensitive indicator of cytotoxicity. However, such techniques may provide important mechanistic data on the role of inflammatory mediators in the irritative response to certain chemicals, or on the possible role and interaction of certain mediators in the irritative process.

Surfactant-induced cytotoxicity in cultures of human keratinocytes and a commercially available cell line (3T3)

Biochemical parameters of cytotoxicity, such as the release of intracellular enzymes, appear to be useful for classification of irritant substances following in vivo chemical insult to the skin. Changes in activity of acid phosphatase (AP), a lysosomal enzyme, appear to parallel the development of the inflammatory response in laboratory animals after treatment with the known skin irritant sodium dodecyl sulphate (SDS). Neutral red (NR) uptake and AP were chosen as endpoints of cytotoxicity. NR and AP were measured in cultures of human epidermal keratinocytes, prepared from separate individuals, and in 3T3 cells following treatment with SDS. The NR(50) value (concentration producing a 50% reduction in NR uptake compared with controls) was similar in both cell types. AP in human keratinocyte cultures exhibited a peak activity, before declining at higher concentrations. This phenomenon was time dependent and was observed within 4 hr of treatment, but was not evident after a 24-hr exposure. The peak produced in 3T3 cells was negligible in comparison. The rate of NR uptake was also studied within the first 4 hr of exposure to SDS, which was comparable to the earlier time points at which AP was determined. The degree of inhibition of NR uptake was greater in human keratinocyte cultures than in 3T3 cells and a response was also elicited at lower dose levels in keratinocytes. AP may be a sensitive indicator of cytotoxicity in human keratinocytes and the data may be interpreted in relation to changes in lysosomal membrane function. This assay may be of value in assessment of skin irritation potential of aqueous soluble surfactants and chemicals that possess the ability to damage biological membranes.

Cytotoxicity of 12 chemicals of known human and animal skin irritation potential in human keratinocyte cultures.

12 chemicals were selected for cytotoxicity testing, based on a report of a collaborative study (EC Project 86E3-063/86-460) that compared in vitro cytotoxicity in animal cell models with animal irritation and non-invasive human in vivo data. Neutral red (NR) and acid phosphatase (AP) activity were chosen as endpoints of cytotoxicity. AP activity has been shown to be a specific indicator of cytotoxicity in keratinocytes. NR(50) values (concentration that produces a 50% reduction in NR uptake compared with controls) and AP((peak)) values (concentration at which a peak activity occurs) were calculated for each chemical. Both in vitro assays exhibited good correlations with the animal irritation data. Poor correlations were obtained for comparison of the cutaneous blood flow volume data. The data presented provide useful information on the relationship between in vivo skin irritation and in vitro experimental models.

Detection of chemical-induced unscheduled DNA synthesis in cultures of normal adult human keratinocytes

Human keratinocytes are the most appropriate target cells to employ in an in vitro model to study the potential ability of a chemical to react with the genome of human skin cells. The resulting damage to DNA may be a key initiating factor in the subsequent development of squamous epithelial carcinoma, which is a common form of cancer in humans. Keratinocytes were routinely established from adult human skin samples and maintained in culture for at least three passages. Genotoxic damage was assessed by a quantitative autoradiographic technique which enables detection of unscheduled DNA synthesis without prior treatment of the cultures with agents, such as hydroxyurea, to suppress S-phase synthesis. Tertiary cultures from two individuals were exposed to the experimental carcinogens, methyl methane sulphonate (direct-acting genotoxin) and benzo[a]pyrene (requiring metabolic activation). Both chemicals induced unscheduled DNA synthesis in human keratinocytes. This study indicates that human keratinocyte cultures are a suitable model for the detection of genotoxic potential in human epidermis.

Prostaglandin E2 release in keratinocyte cultures following exposure to various tumour promoters

The interaction of tumour promoters with the target cell type (keratinocyte) may be an essential feature of their promoting activity and their ability to initiate an inflammatory response. The role of prostaglandin E(2) (PGE(2)), particularly in the keratinocyte, remains largely unknown, but it is closely associated with inflammation and regenerative epidermal hyperplasia, which appear critical for tumour promotion. Rat keratinocytes derived from sublingual mucosa represent a suitable model to investigate the ability of several irritants, of varying tumour-promoting potency, to stimulate PGE(2) release. Cytotoxicity was evaluated by the neutral red uptake assay and a concentration that reduced cell viability to 50% of control was selected as a maximum concentration for subsequent measurement of PGE(2) release. Phorbol-12-myristate-13-acetate, ionophore A23187 and mezerein stimulated PGE(2) release at non-toxic concentrations. Anthralin, benzoyl peroxide, sodium dodecyl sulfate and acetic acid did not stimulate PGE(2) at non-toxic concentrations, but release was associated with a toxic response. Epidermal growth factor and phospholipase C, which are closely associated with intracellular signalling systems that modulate keratinocyte proliferation and differentiation, also stimulated PGE(2) release. Epidermal growth factor elicited PGE(2) release at a concentration reported to be mitogenic in keratinocyte cultures. The stimulation of PGE(2) release in the absence of a toxic response by PMA, mezerein and ionophore A23187 may be indicative of a direct interaction of the chemicals with intracellular pathways involved in regulation of keratinocyte differentiation and proliferation. This interaction may also reflect the ability of such chemicals to initiate an inflammatory response. Measurement of PGE(2) release may be useful to investigate further the mechanism of action of tumour promoters in the target cell type. In contrast, other tumour promoters did not stimulate release at non-toxic concentrations, which implies that their ability to initiate an inflammatory response and possibly their promoting activity is associated with the induction of a toxic response in the target cell population.

Measurement of eicosanoid release in keratinocyte cultures to investigate skin irritation and tumour promoting activity

Skin irritation, inflammation and hyperplasia appear to be intimately associated with the phenomenon of tumour promotion, but the mechanism of action remains elusive. Prostaglandins and leukotrienes play an important role in skin inflammation and prostaglandin E(2) (PGE(2)) modulates several events associated with phorbol ester-induced tumour promotion. This study investigated the release of eicosanoids (PGE(2) and leukotriene B(4)) and markers of cytotoxicity [neutral red (NR) uptake and intracellular acid phosphatase (AP) activity], after exposure of rat tongue epithelial (RTE) keratinocyte cultures to chemicals of different irritating and tumour promoting activity. The potent phorbol ester tumour promoter phorbol-12-myristate-13-acetate (PMA), and the less potent, structurally related diterpene ester, mezerein (MEZ) were compared with the known skin irritant sodium dodecyl sulfate (SDS). Cytotoxicity data reflected the in vivo skin irritation potential of the test chemicals and intracellular AP activity was increased after exposure to SDS (160 mug/ml), but did not appear to be increased by the more cytotoxic chemicals PMA and MEZ. Extracellular levels of PGE(2) were increased (200 to > 1000% of control levels) after an 18-hr exposure to PMA or MEZ over a concentration range of 0.01 to 20 mug/ml (NR(50) values 8.0 +/- 6.6 and 15.5 +/- 4.8 mug/ml, respectively). These data indicated that PGE(2) release occurred in the absence of cytotoxicity. In contrast, SDS only elicited PGE(2) release after exposure to 80 mug/ml (a cytotoxic dose level, NR(50) 82.5 +/- 9.9 mug/ml). The potency of a chemical to elicit PGE(2) release in keratinocytes in the absence of a cytotoxic response may reflect intracellular pathways intimately associated with the initiation of an inflammatory response and possibly with tumour promoting activity.

Chemically-induced unscheduled DNA synthesis in cultures of adult human epidermal keratinocytes

Skin is a major target organ for many experimental carcinogens that exist in our environment and the majority of previous carcinogenicity studies have utilised animal derived models. In view of the fact, that many of these environmental chemicals exhibit species- and tissue-specific metabolism, a human skin tissue derived model would be a distinct advantage. Squamous epithelial carcinoma is a predominant form of skin cancer in man and, in theory, human epidermal keratinocytes present an appropriate target cell to employ as an in vitro system to study epidermal carcinogenesis. This report demonstrates the valuable potential of human keratinocyte cultures as a suitable model for mechanistic studies on factors which may influence DNA damage and, hence, the subsequent development of cancer in human epidermis. Keratinocytes were serially cultivated from adult human skin samples and maintained in culture for at least 3 passages. Tertiary cultures, isolated from 3 separate individuals, were exposed to the direct-acting experimental carcinogen, methyl methanesulphonate (CAS No. 66-27-3), and benzo[a]pyrene (CAS No. 50-32-8), which requires metabolic activation. DNA repair was assessed by a quantitative autoradiographic technique. Methyl methanesulphonate and benzo[a]pyrene both elicited a dose-related increase in unscheduled DNA synthesis in cultures prepared from each individual. Inter-individual variation in the response was observed for each chemical, but this was greater in the case of benzo[a]pyrene, which indicates inter-individual variation in both xenobiotic metabolism activity and DNA repair capacity.