Jean-Claude Ourlin

Human Hepatocyte Culture

Primary culture of human hepatocytes is an in vitro model widely used to investigate numerous aspects of liver physiology and pathology. The technique used to isolate human hepatocytes is based on two-step collagenase perfusion. Originally performed in situ for obtaining hepatocytes from the adult rat, this technique has been adapted to the ex vivo treatment of human liver from organ donors or from lobectomy resection for medical purposes. This chapter describes experimental protocols for the isolation of hepatocytes from human liver tissue and for the preparation of short- and long-term cultures in which cells retain a differentiated phenotype for at least 1 mo. The various aspects emphasized here include the conditions for obtaining tissue, quality control of tissue for efficient perfusion, collagenase perfusion parameters, solutions for perfusion and culture media, cell substrate, cell plating, specific equipment, and safety conditions.

HMOX1 and NQO1 Genes are Upregulated in Response to Contact Sensitizers in Dendritic Cells and THP-1 Cell Line: Role of the Keap1/Nrf2 Pathway

Electrophilicity is one of the most common features of skin contact sensitizers and is necessary for protein haptenation. The Keap1 (Kelch-like ECH-associated protein 1)/Nrf2 -signaling pathway is dedicated to the detection of electrophilic stress in cells leading to the upregulation of genes involved in protection or neutralization of chemical reactive species. Signals provided by chemical stress could play an important role in dendritic cell activation and the aim of this work was to test whether contact sensitizers were specific activators of the Keap1/Nrf2 pathway. CD34-derived dendritic cells (CD34-DC) and the THP-1 myeloid cell line were treated by a panel of sensitizers (Ni, 1-chloro 2,4-dinitrobenzene, cinnamaldehyde, 7-hydroxycitronellal, 1,4-dihydroquinone, alpha-methyl-trans-cinnamaldehyde, 2-4-tert-(butylbenzyl)propionaldehyde or Lilial, and 1,4-phenylenediamine), irritants (sodium dodecyl sulfate, benzalkonium chloride), and a nonsensitizer molecule (chlorobenzene). Three well-known Nrf2 activators (tert-butylhydroquinone, lipoic acid, sulforaphane) were also tested. Expression of hmox1 and nqo1 was measured using real-time PCR and cellular accumulation of Nrf2 was assessed by Western blot. Our results showed an increased expression at early time points of hmox1 and nqo1 mRNAs in response to sensitizers but not to irritants. Accumulation of the Nrf2 protein was also observed only with chemical sensitizers. A significant inhibition of the expression of hmox1 and nqo1 mRNAs and CD86 expression was found in 1-chloro 2,4-dinitrobenzene-treated THP-1 cells preincubated with N-acetyl cysteine, a glutathione precursor. Altogether, these data suggested that the Keap1/Nrf2-signaling pathway was activated by electrophilic molecules including sensitizers in dendritic cells and in the THP-1 cell line. Monitoring of this pathway may provide new biomarkers (e.g., Nrf2, hmox1) for the detection of the sensitization potential of chemicals.

SENS-IS, a 3D reconstituted epidermis based model for quantifying chemical sensitization potency: Reproducibility and predictivity results from an inter-laboratory study.

The SENS-IS test protocol for the in vitro detection of sensitizers is based on a reconstructed human skin model (Episkin) as the test system and on the analysis of the expression of a large panel of genes. Its excellent performance was initially demonstrated with a limited set of test chemicals. Further studies (described here) were organized to confirm these preliminary results and to obtain a detailed statistical analysis of the predictive capacity of the assay. A ring-study was thus organized and performed within three laboratories, using a test set of 19 blind coded chemicals. Data analysis indicated that the assay is robust, easily transferable and offers high predictivity and excellent within- and between-laboratories reproducibility. To further evaluate the predictivity of the test protocol according to Cooper statistics a comprehensive test set of 150 chemicals was then analyzed. Again, data analysis confirmed the excellent capacity of the SENS-IS assay for predicting both hazard and potency characteristics, confirming that this assay should be considered as a serious alternative to the available in vivo sensitization tests.

Evidence for chemical and cellular reactivities of the formaldehyde releaser bronopol, independent of formaldehyde release.

Formaldehyde and formaldehyde releasers are widely used preservatives and represent an important group of skin sensitizers. Formaldehyde is very often suspected to be the sensitizing agent of formaldehyde-releasers; however, many reported clinical cases of contact allergy to these molecules such as bronopol (2-bromo-2-nitropropane-1,3-diol) indicate negative skin reactions to formaldehyde suggesting a more complex mechanism. The aim of this study was to compare the chemical reactivity and biological activity of formaldehyde with those of two formaldehyde releasers: 2-bromo-2-nitropropane-1,3-diol and 1,3-dimethylol-5,5-dimethylhydantoin. A key step in the sensitization to chemicals is the formation of the hapten-protein antigenic complex via covalent binding between the chemical sensitizer and amino acids in proteins. The chemical reactivity of the three compounds was thus addressed using (13)C NMR analysis of adduct formation upon incubation with a set of nucleophilic amino acids. The biological activity was measured in two in vitro models based on dendritic cells and a monocytic cell line (CD34-DC and THP-1 model) through monitoring of a panel of biomarkers. The results obtained show that 2-bromo-2-nitropropane-1,3-diol produces low amount of free formaldehyde in physiological buffers but that its degradation generates various molecules including 2-bromoethanol. In addition, 2-bromo-2-nitropropane-1,3-diol also generates adducts with amino acids, not observed with formaldehyde alone, that could be explained by the reactivity of 2-bromoethanol. In parallel, in a cellular approach using the human monocytic THP-1 cell line, 2-bromo-2-nitropropane-1,3-diol activates THP-1 cells at concentrations that are not correlated to simple formaldehyde release. This observation is confirmed in the more physiological model CD34-DC. Moreover, in the THP-1 model, the expression profiles of several biomarkers are specific to 2-bromo-2-nitropropane-1,3-diol. Finally, the use in the cellular model of the pure degradation products identified by NMR reveals the reactivity of bromonitromethane. In contrast, 1,3-dimethylol-5,5-dimethylhydantoin presents chemical and biological reactivities similar to those of formaldehyde. Taken together, these data suggest that 2-bromo-2-nitropropane-1,3-diol is an atypical formaldehyde releaser, releasing low amounts of formaldehyde at physiological conditions but producing multiple degradation products among which 2-bromoethanol and bromonitromethane are potential candidates for explaining the specific allergic reactions to 2-bromo-2-nitropropane-1,3-diol.