Generation of oxidative stress in human cutaneous models following in vitro ozone exposure.

Ozone, one of the main components of photochemical smog, represents an important source of environmental oxidative stress. The skin, being the outermost barrier of the body, is directly exposed to environmental oxidant toxicants. Skin sebum and cellular plasma membrane lipids contain polyunsaturated fatty acids which are primary targets for ozone and free radical attack induced lipid peroxides. These ozonation processes in skin can also generate aldehydes, hydroxyhydroperoxides and specific Criegee's ozonides. In order to evaluate in vitro human skin susceptibility to ozone, we have exposed cultured immortalized human keratinocytes (DK7-NR) and the reconstructed human epidermis Episkin to 10 ppm of ozone in a specific incubator. We measured the formation of protein carbonyls by an ELISA method and monitored the oxidative stress using the fluorogenic probe 2',7'-dichlorofluorescin-diacetate (DCFH-DA). Results showed a time-dependent increase of fluorescence levels (linked to oxidative stress) in both models exposed to ozone. Using this protocol, we investigated the protective potential of different products including vitamin C, a thiol derivative and a plant extract. All products dramatically reduced oxidative responses during ozone exposure. Decreases observed in fluorescence levels were between 60 and 90% as compared to non-protected controls. These results demonstrate: (a) cutaneous in vitro models are remarkably susceptible to oxidative stress generated by an environmental air pollutant as ozone, and (b) raw antioxidants, thiols and vitamin C were efficient products to prevent ozone induced cellular oxidative damage.

Evaluation of eye irritation potential: statistical analysis and tier testing strategies.

Eye irritation testing, specifically the Draize test, has been the centre of controversy for many reasons. Several alternatives, based on the principles of reduction, refinement and replacement, have been proposed and are being used by the industry and government authorities. However, no universally applicable, validated non-animal alternative(s) is currently available. This report presents a statistical analysis and two testing approaches: the partial least squares multivariate statistical analysis of de Silva and colleagues from France, the tier-testing approach for regulatory purposes described by Gerner and colleagues from Germany, and the three-step tier-testing approach of the US Interagency Regulatory Alternatives Group described by Gupta and Hill. These approaches were presented as three separate papers at the November 1993 Interagency Regulatory Alternatives Group (IRAG) Workshop on Eye Irritation Testing; they have been summarized and combined into the following three-part report. The first part (de Silva et al.) presents statistical techniques for establishing test batteries of in vitro alternatives to the eye irritation test. The second (Gerner et al.) and third (Gupta and Hill) parts are similar in that they stage assessment of information by using a combination of screening information and animal testing to effect reductions in animal use and distress.

The use of in vitro methods in the ocular irritation assessment of cosmetic products.

The ocular tissue is a complex system consisting of corneal and conjunctival epithelial cells, the underlying corneal stroma and associated endothelial cells. Exposure to chemicals may result in responses ranging from mild, slight redness and itching, to severe injury with loss of corneal epithelium, damage to stroma, inflammatory infiltration and loss of vision. This complexity hinders the development of in vitro methods able to replace animal testing. Various in vitro techniques have been proposed and subsequently developed as potential replacements for ocular toxicity screening on animals. Over the past 2 years, eight methods have been evaluated in these laboratories. The endpoint of these methods could be linked to one or to several clinical events occurring in the in vivo eye irritancy process described above. Using these systems, a battery of four complementary in vitro assays has been developed. For the categories of ingredients and cosmetic products investigated, the promising results obtained suggest that in vitro methods of ocular risk assessment may be used increasingly in the future.

The silicon microphysiometer for testing ocular toxicity in vitro.

The silicon microphysiometer has been used for in vitro evaluation of the ocular irritancy potential of water soluble ingredients and formulations. This light-addressable potentiometric sensor detects changes in cell physiology by monitoring the rate at which cultured cells excrete their acidic products of metabolism. We have mainly determined the metabolic effect of 53 products (21 surfactants and 32 surfactant-based formulations). The related maximal average Draize score (MAS) were available from historical data and varied from 1.7 to 54. All of the Draize categories were represented. Murine fibroblastic cells (L929 clone) were exposed to increasing concentrations of the product for approximately 400 sec per dose. The MRD(50) (dose of product that decreased the metabolic rate of the cells by 50%) was determined by interpolation from a plot of metabolic rate versus test material concentration. Decreases in metabolic rate, as assessed by the MRD(50), occurred over a wide range of concentrations (40 mug/ml-200 mg/ml). The linear (Pearson) and rank (Spearman) correlation between in vivo (MAS) and in vitro (log MRD(50)) data were 0.91 and 0.89, respectively. This study indicates that the silicon microphysiometer method exhibits a high correlation with the Draize test for water-soluble raw materials and formulations and thus can be used as an in vitro screen for ocular irritation.

In vitro methods: their relevance and complementarity in ocular safety assessment.

Ocular irritation includes a wide variety of mechanisms some of which can be explored by in vitro methods. For example, the effects on epithelial cells that constitute the outer layers of both the conjunctiva and the cornea may result in direct cytotoxicity or impairment of cellular functions -such as impermeability-, phenomena that can be explored in vitro. Irritancy may also involve inflammation of the conjunctival connective tissue and of the corneal stroma with its vascular and cellular features; effects on the stroma can lead to the opacification of the cornea; this last phenomenon may be the consequence of mechanisms such as modification of the structure of proteins or changes in stroma hydration which in particular is closely related to corneal endothelium metabolic activity. Recovery after eye injury depends partly on the extent of ocular damage and on the residual mitotic activity of the remaining cells. We have studied 41 surfactants, lotions and shampoos in 6 to 8 in vitro methods each one exploring one or two endpoints that could be linked to the ocular irritancy phenomena described above. In vivo ocular irritancy data for these materials from previous studies were compared to in vitro results. The results obtained show that -among the techniques that were investigated and for the categories of substances that were studied- the Het-CAM test and more particularly the endpoint that is related to vascular effects gives the best assessment of acute ocular irritancy (Spearman's rho coefficients between in vivo and in vitro data greater than 0.90); however, cell culture methods, especially one based on short contact time between cells and products and on evaluation of early toxic effects, also proved interesting (Spearman's rho coefficients between in vivo and in vitro data greater than 0.85). Moreover, the isolated cornea opacity and permeability test gave complementary information more related to recovery from surfactant-induced damage. These encouraging results lead us to consider in vitro ocular safety assessment with optimism for the categories of products investigated.

Development of mechanisms of protection against oxidative stress in doxorubicin-resistant rat tumoral cells in culture.

We have compared some mechanisms involved in the defense against doxorubicin-induced free radical damage in rat hepatoma and glioblastoma cell lines and their doxorubicin-resistant variants presenting an overexpression of the multidrug resistance gene. Immediate in vivo production of malondialdehyde was minor and was not different in sensitive and resistant cells. Alpha-tocopherol was undetectable in all cell lines. Glutathione levels were not different in sensitive and resistant cells and these levels did not vary upon doxorubicin treatment. Resistant cells exhibited either a 50% decrease (hepatoma) or a 25% increase (glioblastoma) of glutathione-S-transferase activity. Glutathione reductase presented no important change upon acquisition of resistance. In contrast, selenium-dependent glutathione peroxidase activity was consistently 2-6-fold increased in the resistant cells, which suggests a magnification of protection mechanisms against hydroxyle radical formation from H2O2 in resistant cells. Depletion of glutathione levels by buthionine sulfoximine sensitized hepatoma resistant cells to doxorubicin, but had no effect on doxorubicin cytotoxicity to glioblastoma cells.

[Circadian variation of the nephrotoxicity induced in rats by bi-daily repeated administration of amikacin]. / Variations temporelles de la néphrotoxicité induite chez le rat par administration bi-quotidienne réitérée d'amikacine.

We have studied the chrononephrotoxicity of amikacin during and after a prolonged treatment in rats. Animals received by intramuscular route a daily dose of 400 mg/kg (in two times) during 7 days at two different times (8.00 - 20.00 and 14.00 - 2.00). Large time-dependent variations in renal injury had been evidenced by several parameters and particularly by enzymuria. This injury is maximal when administration is made at 14.00 - 2.00. These data confirmed our results obtained in previous investigations with single daily injections and evidence that chrononephrotoxicity could permit an optimization of nephrotoxic drugs largely used in clinics.

[Demonstration of seasonal changes of circadian rhythms of amikacin chrononephrotoxicity in rats]. / Mise en évidence des variations saisonnières des rythmes circadiens de la chrononéphrotoxicité de l'amikacine chez le rat.

The present study investigates the chronobiological approach of the amikacin-induced nephrotoxicity in rats treated with a single sublethal dose administered at different times of day. The nephrotoxicity is appreciated by gamma-glutamyl-transferase and N-acetyl-beta-D-glucosaminidase urinary excretion, respectively a brush border and a lysosomal enzyme. These excretions peak out at 14:00 and reach a through at 20:00. In contrast, in a precedent experimentation, in october/november, we evidence gamma-glutamyl-transferase excretion increased at 20:00. So, we evidenced with amikacin not only a circadian but also a seasonal susceptibility in rats.

[Chrononephrotoxicity of amikacin after 7-day chronic poisoning in rats]. / Etude de la chrononéphrotoxicité de l'amikacine après intoxication chronique de sept jours chez le rat.

We have studied the chrononephrotoxicity of amikacin during and after a prolonged treatment in rats. Animals received by intramuscular route a daily dose of 400 mg/kg during 7 days at four different times (08:00, 14:00, 20:00 or 02:00). Large time-dependent variations in renal injury had been evidenced by several parameters and particularly by enzymuria. This injury is maximal when administration is made at 14:00. These data confirmed results obtained in previous investigations. The knowledge of chrononephrotoxicity could permit an optimization of drugs in clinics.

[Importance of urinary enzymes as a marker of post-ischemic proximal tubular involvement in rat]. / Intérêt de l'enzymurie comme marqueur de l'atteinte tubulaire proximale post-ischémique chez le rat.

We have studied in rats the influence of renal ischemia on urinary excretion of three brush border membrane enzymes (gamma glutamyl transferase, alkaline phosphatase and leucine aminopeptidase) and of a lysosomal one (N-acetyl-B-D-glucosaminidase). Urines were collected over 24 hours periods during three days before and after a 45 minutes renal artery clamping. Urinary GGT, PAL and LAP excretion were significantly increased on the first day after renal ischemia, but returns to normal values on the second day. Urinary NAG activity increases on the first day, but contrary to the latter enzymes, reached to normal values only on the third day. Enzymuria seems to be a useful marker of tubular injury occurring after a temporary renal ischemia in the rat.