Serum microRNA biomarkers for drug-induced liver injury.

New biomarkers of drug-induced liver injury (DILI) are required in the clinic and in preclinical pharmaceutical evaluation. Liver-enriched microRNAs are promising serum biomarkers of acetaminophen-induced acute liver injury in mice. The utility of circulating microRNAs as biomarkers of human acute DILI is discussed in the context of correlation with existing biomarkers of liver injury and patient outcomes in acetaminophen toxicity, mechanisms of cellular microRNA release, and their potential advantages over current clinical biomarkers of DILI.

Cytokine expression profiles during murine contact allergy: T helper 2 cytokines are expressed irrespective of the type of contact allergen.

We have investigated the cytokine response pattern following sensitisation (induction) of BALB/c mice with different chemicals (dinitrochlorobenzene, dinitrofluorobenzene, oxazolone, glutaraldehyde, formaldehyde, trimellitic anhydride, croton oil) and elicitation (challenge) of contact allergy in sensitised animals. The results of our investigations showed that different chemicals induced both T helper (Th) 1 cytokines [interleukin (IL) 2, interferon beta (IFNgamma) [corrected] and Th2 cytokines (IL-4, IL-10) at different stages during murine contact allergy. We also confirmed our previous findings that IL-4 and IL-10 release were up-regulated during the challenge phase regardless the contact allergen used, whereas the release of IFNgamma [corrected] did not show a clear preference for being up- or down-regulated. In our hands, the increased expression of Th2 cytokines after challenge exposure to contact allergens appeared as a stable marker of secondary contact allergenic responses. Quantitative differences in the expression of IL-4 were observed between different contact allergens. The present results clearly indicate that skin sensitisers were able to elicit cytokine response patterns, which could not be related to a clear-cut Th1 or Th2 type of cytokine response. Furthermore, dermal application of contact allergens produced different kinetics of cytokine secretion upon induction and challenge. In our hands, the co-expression of Th1 and Th2 type cytokines appeared as a universal consequence of dermal application of contact allergens to responsive mice. Our results indicate that co-expression of Th1 and Th2 cytokines during contact allergy is an important feature of murine contact allergy in responsive mice and that chemicals differ in their potency to induce the expression of these cytokines. Furthermore, the results do not support the view that different chemicals induce Th1 or Th2 cytokines in a mutually exclusive manner depending on their preference for inducing either contact or respiratory allergy. The results are expected to renew the discussion about the usefulness of the Th1/Th2 paradigm in certain areas of immunotoxicology.

Assessment of the phototoxic potential of compounds and finished topical products using a human reconstructed epidermis.

The goal of this study was to design a model system for the assessment of phototoxic potential using a human reconstructed epidermis (HRE, SkinEthic Laboratories, Nice, France), by testing some representative phototoxic (P) and non-phototoxic (NP) compounds and finished topical products. The tissue response to 24-h application of 5-5000 microg/mL of the test agents in the presence and absence of UVA light was analyzed in terms of viability (Lactate Dehydrogenase release), pro-inflammatory activity (IL-8 release and mRNA expression) and morphology (histopathology). 8-Methoxypsoralen (P) and promethazin (P), but not sodium lauryl sulfate (NP) produced cytotoxicity concentration-response curves significantly different between irradiated and nonirradiated tissues. Only irradiated tissues showed morphological damage. Application of tetracyclin (P) in the culture medium, but not topically, induced similar signs of phototoxicity. 6-Methylcoumarine (weak P) was not cytotoxic, yet it increased IL-8 release and mRNA expression only following UVA irradiation. PUVA therapy creams containing 1% 8-Methoxy-psoralen (P) or coal tar (P) decreased viability and induced histologic damage in UVA-exposed tissues. In conclusion, the phototoxic potential of the tested agents was correctly predicted by using a tiered strategy that involves determining cytotoxicity, production of IL-8, and morphological damage following exposure of the HRE to the compounds and UVA light.

Evidence for the impairment of the vitamin D activation pathway by cyclosporine A.

Cyclosporine A (CsA) is a potent immunosuppressant with the drawback of renal side effects. We reported that CsA markedly decreases calcium-binding protein calbindin-D28k mRNA levels in rat kidneys, and showed that this decrease is associated with its adverse renal effects. The transcription of the calbindin-D28k gene is activated via the vitamin D pathway. In this work, the potential CsA-mediated impairment of the vitamin D pathway was investigated. Wistar rats were treated for 12 days with 50 mg/kg/day CsA or for 20 days with 50 mg/kg/day of the non-immunosuppressant and non-nephrotoxic SDZ PSC 833, which had been previously shown not to affect calbindin-D28k mRNA levels. The expression of the three vitamin D-regulated genes calbindin-D28k, 1,25-dihydroxyvitamin D3-24-hydroxylase (24-OHase), and vitamin D receptor (VDR) were quantified in rat kidney homogenates by real-time reverse transcription-polymerase chain reaction. Plasma parathyroid hormone (PTH) as well as plasma and kidney 1,25 dihydroxyvitamin D3 (calcitriol) levels were monitored in all animals. CsA induced a 85% decrease in calbindin-D28k mRNA levels as well as a 40% and 69% decrease in VDR and 24-OHase mRNA levels, respectively. Plasma and kidney 1,25 dihydroxyvitamin D3 as well as plasma PTH levels were increased by CsA, but not by SDZ PSC 833. The treatment with SDZ PSC 833 did not affect calbindin-D28k or VDR expression, but did cause a 73% decrease in 24-OHase mRNA levels. Taken together, these results indicate an association between CsA-mediated down-regulation of rat renal calbindin-D28k mRNA and the decrease in other 1,25 dihydroxyvitamin D3-regulated genes, suggesting an impairment of the vitamin D pathway by CsA which may be related to its adverse renal side effects and its immunosuppressive activity.

New insights into cyclosporine A nephrotoxicity by proteome analysis.

Using two-dimensional gel electrophoresis (2-DE), we recently discovered an association between decreased calcium-binding protein, calbindin-D 28 kDa, urinary calcium wasting and intratubular corticomedullary calcifications in rat kidney. This observation prompted us to investigate kidney tissues of other species, including man. In this paper we show that in dogs and monkeys, which are generally devoid of cyclosporine A (CsA)-mediated nephrotoxicity, renal calbindin levels were not affected by the CsA treatment whereas in CsA-treated human kidney-transplant recipients with renal vascular or tubular toxicity, a marked decrease in renal calbindin-D 28 kDa protein level was found in most of the kidney biopsy sections. The present results strongly suggest that calbindin is a marker for CsA-nephrotoxicity. The discovery of calbindin-D 28 kDa being involved in CsA toxicity has evolved from the application of 2-DE and has not been reported previously, proving that proteomics can provide essential information in mechanistic toxicology. Considering the current improvements in proteome methods it is expected that high throughput proteomics will become an indispensable tool in preclinical safety testing.

Cyclosporine A-induced decrease in calbindin-D 28 kDa in rat kidney but not in cerebral cortex and cerebellum.

Recently, we reported that in rat, cyclosporine A (CsA) markedly decreases the levels of calbindin-D (CABP-D) 28 kDa in kidney. CABP-D 28 kDa is a calcium-binding protein which is highly expressed in calcium-transporting tissues such as kidney or brain. In this study, we investigated whether, in addition to the kidney, CsA also has an effect on CABP-D 28 kDa in rat brain. Three groups of male Wistar rats received 15 mg/kg/day or 50 mg/kg/day of CsA orally for 12 days, whereas controls received vehicle solution for the same period. CABP-D 28-kDa protein and CsA were quantified in homogenates of kidney, cerebral cortex and cerebellum, and the localization of CABP-D 28 kDa was assessed in the different tissue sections by immunohistochemistry. In kidney, CABP-D 28 kDa was strongly and dose dependently decreased, and was located in tubular epithelial cells. In brain, CABP-D 28 kDa was not changed and was mainly located in pyramidal cells of the cortex and in cerebellum exclusively in Purkinje cells. High CsA concentrations were measured in kidney, more than 17-fold greater than those found in cortex. In cerebellum, CsA was below the limit of detection. These data suggest that at clinically relevant doses, CsA may not affect CABP-D 28-kDa levels in brain.

The cyclosporine A-induced decrease in rat renal calbindin-D28kDa protein as a consequence of a decrease in its mRNA.

Cyclosporine A (CsA) is a potent immunosuppressant with the drawback of renal side-effects. We recently reported that relatively high doses of CsA markedly decreased the calcium-binding protein calbindin-D28kDa in kidneys of male Wistar rats, and showed that this decrease could be associated with some of the drug-induced adverse renal effects. To investigate the events leading to this decrease, the calbindin-D28kDa mRNA level in kidneys of rats treated with 15 or 50 mg/kg/day CsA for 12 days was analysed by reverse transcription followed by polymerase chain reaction. At both doses, a marked dose-dependent decrease in the calbindin-D28kDa mRNA level was found, one very similar to the decrease measured in the calbindin-D28kDa protein abundance. Thus, the CsA-mediated down-regulation of the renal calbindin-D28kDa protein is most likely the result of a decrease in the calbindin-D28kDa mRNA level.