Development of a screening method for phthalate esters in polymers using a quantitative database in combination with pyrolyzer/thermal desorption gas chromatography mass spectrometry.

Seven phthalate esters (di-isobutyl phthalate (DIBP), di-n-butyl phthalate (DBP), benzylbutyl phthalate (BBP), di-(2-ethylhexyl) phthalate (DEHP), di-n-octyl phthalate (DNOP), di-isononyl phthalate (DINP) and di-isodecyl phthalate (DIDP)) were analyzed by pyrolyzer/thermal desorption-gas chromatography/mass spectrometry (Py/TD-GC/MS), the retention index and relative response factor (RRF) relative to DEHP was calculated for each compound and used to construct a quantitative database (qDB). This qDB enables normalization of the retention time and response factor of each phthalate ester between any laboratory simply by analyzing an n-alkane solution and DEHP standard material. This allows for easy calculation of the phthalate ester content of samples without preparation of calibration curves. The efficacy of this qDB method was verified by performing a quantitative analysis of phthalate esters at 4 different laboratories that showed actual retention times were within ±0.012 min of the estimated retention times for all compounds at all laboratories. Similarly, the mean recovery rate (n = 6) at each laboratory was within 79-113%. Quantitative analysis was also performed on 30 real samples using both the qDB method and the Py/TD-GC/MS method set forth in IEC62321-8, which involves the preparation of 1-point calibrations to perform quantitative analysis. The difference in quantitative results between the methods was approximately within ±200 mg/kg for compounds in the concentration region of <2000 mg/kg.

Regulation of matrix metalloproteinase-1 and alpha-smooth muscle actin expression by interleukin-1 alpha and tumour necrosis factor alpha in hepatic stellate cells.

Hepatic stellate cells (HSCs) are key players in liver fibrosis and regeneration via collagen degradation and synthesis. These phenomena involve inflammatory cytokines released from non-parenchymal liver cells such as Kupffer cells. Although the effects of individual cytokines on many cell types have been investigated in various conditions, such as inflammation and tissue fibrosis, investigating the effect of combined cytokines would further our understanding of the regulatory mechanisms in tissue fibrosis. Here, we report the effect of multiple cytokine combinations on primary HSCs. We first examined the effect of individual cytokines and then the simultaneous exposure of different cytokines, including interleukin-6 (IL-6), IL-1 alpha (IL-1α), platelet-derived growth factor (PDGF), tumour necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-ß), on matrix metalloproteinase-1 (MMP1) gene expression in primary HSCs. We observed that the combination of all five cytokines induced higher levels of MMP1 gene expression. Of these cytokines, TNF-α and IL-1α were found to be the key cytokines for not only inducing MMP1 expression, but also increasing α-smooth muscle actin gene expression. In conclusion, the combined treatment of TNF-α and IL-1α on HSCs had an enhanced effect on the expression of the fibrotic genes, MMP1 and α-smooth muscle actin, so appears to be an important regulator for tissue regeneration. This finding suggests that stimulation with combined anti-fibrotic cytokines is a potential approach in the development of a novel therapy for the recovery of liver fibrosis.

Human primary cultured hepatic stellate cells can be cryopreserved.

We compared the morphological and functional characteristics of cultured unfrozen hepatic stellate cells (HSCs) and cryopreserved HSCs obtained from human livers. We used liver tissues obtained by surgical resection from patients with metastatic liver cancer or with hepatocellular carcinoma. HSCs were isolated and allowed to spread in culture. Comparison of morphological and functional features between the unfrozen HSCs and cryopreserved HSCs was performed at each passage using the following techniques: light microscopy, immunohistochemistry, cell growth curve, metallothionein (MTT) assay, and PI staining, Western blot, real-time polymerase chain reaction (PCR), and gene expression analysis using microarrays. The purity of HSCs was more than 90% in all passages. alpha-Smooth muscle actin (SMA-)positive HSCs gradually increased in successive passages, and the positive cell rate and rate of increase in cell number were similar in both groups. Expression of platelet-derived growth factor (PDGF) receptor, transforming growth factor (TGF)-beta receptor, and alpha-SMA mRNAs and protein was similar during each passage in the two groups. Gene expression was nearly identical at each passage in unfrozen and frozen/thawed samples obtained from the same patient. In conclusion, an adequate protocol for the cryopreservation of human primary cultured HSCs could be established.