M.H. Grant

Interactions of orthopaedic metals with an immortalized rat osteoblast cell line

The toxicity of nickel, chromium (III) and (VI), vanadium and aluminium was compared in an immortalized neonatal rat osteoblast cell line using the MTT assay and a novel index of cytotoxicity, alkaline phosphatase (ALP) activity. Where toxicity was observed, ALP was a consistently more sensitive detection method than the MTT assay. The toxicity of the metals increased in the order aluminium < chromium (III) < vanadium < nickel < chromium (VI). alpha-Tocopherol partially prevented nickel-induced toxicity (as assessed by ALP activity), whereas ascorbic acid had no protective effect. Chromium (VI) was more toxic than (III), with significant toxicity observed at 0.5 microM. It is thought that Cr (III) cannot readily penetrate the cell membrane and this may account for the lower toxicity. Aluminium had a stimulatory effect on cell growth at low concentrations (0.5 microM). The combination of immortalized rat osteoblasts and the ALP activity test provides a powerful tool for in vitro testing of orthopaedic materials.

Investigation into the biological stability of collagen/chondroitin-6-sulphate gels and their contraction by fibroblasts and keratinocytes: the effect of crosslinking agents and diamines

Artificial skin substitutes based on autologous keratinocytes cultured on collagen-based substrata are being developed for grafting onto patients with severe burns. The properties of the substratum can be manipulated by crosslinking the collagen with the glysocaminoglycan, chondroitin-6-sulphate (Ch6SO4), carbodiimides and polyamines. Biological stability, assessed by resistance to collagenase, was increased by incorporation of Ch6SO4, but crosslinking with the carbodiimides, 1-ethyl-3-(dimethylaminopropyl)carbodiimide and 1,1-carbonyldiimidazole or the polyamines, putrescine or diaminohexane, had little further benefit. Contraction of the collagen gels occurred to a greater extent when seeded with fibroblasts than with keratinocytes. The extent of contraction by either cell type was not influenced by the presence of Ch6SO4 in the gel, but the carbodiimides, and to a lesser extent the polyamines, limited cell-mediated contraction, particularly that mediated by fibroblasts. Optimum substratum composition for artificial skin substitutes will involve a compromise between the desired attributes of biological stability, rate of contraction, mechanical strength, biocompatibility and promotion of cell growth.

Metabolism of quercetin and kaempferol by rat hepatocytes and the identification of flavonoid glycosides in human plasma

1. The metabolism of the flavonoids quercetin and kaempferol by rat hepatocytes was investigated using liquid chromatography coupled with electrospray mass spectrometry (LC-ESI MS). Quercetin and kaempferol were extensively metabolized (98.8 ± 0.1% and 81.0 ± 5.1% respectively, n = 4), with four glucuronides of quercetin and two of kaempferol being detected after incubation. 2. The glucuronides of quercetin and kaempferol formed upon incubation with rat hepatocytes were identified as the same ones formed after incubation with the UDP-glucuronosyltransferase isoform UGT1A9. 3. In addition, plasma samples from human volunteers taken after consumption of capsules of Ginkgo biloba, a plant rich in flavonoid glycosides, were analysed by LC-MS for the presence of flavonoid glucuronides and flavonoid glycosides. Reported is evidence for the presence of flavonoid glycosides in samples of plasma. 4. The results suggest that UGT1A9 is a key UDP-glucuronosyltransferase isoform for the metabolism of flavonoids, and that absorption of intact flavonoid glycosides is possible.

The response of primary rat and human osteoblasts and an immortalized rat osteoblast cell line to orthopaedic materials: comparative sensitivity of several toxicity indices

When studying the biocompatibility of orthopaedic biomaterials it isoften necessary to discriminate between responses which show mild cytotoxicity.It is therefore essential to use a very sensitive index of toxicity. We havecompared the sensitivity of four well-established indices of toxicity: totalcell protein content, leakage of lactate dehydrogenase (LDH), reducedglutathione content and the MTT assay, with that of a novel index, alkalinephosphatase (ALP) activity. Comparisons were made by detecting nickel chloridetoxicity in osteoblasts. ALP activity, the novel method, proved the mostsensitive index of toxicity and it provides a convenient automated assay forassessing the interactions of materials with osteoblasts. The responses tonickel chloride and to aqueous extracts prepared from carbon fibre reinforcedepoxy and polyetheretherketone (peek), two candidate materials for orthopaedicimplants, were compared in primary and immortalized rat osteoblasts, and !in primary human osteoblasts. Although the immortalized rat osteoblast cell line,FFC, was consistently the most sensitive cell type, the responses of the humancells and the FFC cell line were similar in terms of ALP activity throughout therange of nickel concentrations studied. Neither peek nor epoxy material extractsshowed a significant decrease in the MTT or ALP responses in any of the threecell types. Our data suggest that immortalized rat osteoblasts may provide anin vitro model system for screening the biocompatibility of orthopaedicpolymers.

Variation in drug-metabolizing enzyme activities during the growth of human Hep G2 hepatoma cells

1. The activities of several drug-metabolizing enzymes change during the growth cycle (exponential growth to confluence) of Hep G2 cells in culture. As the rate of cell growth slowed down (days 7 to 10 after passage) the activities of ethoxy- and methoxy-resorufin O-dealkylase and of NADPH cytochrome c- and NADH cytochrome b5-reductase increased. In contrast, the O-dealkylations of pentoxy- and benzyloxy-resorufin did not change significantly during culture. 2. UDP-glucuronyltransferase activities also showed substrate-dependent alterations with time in culture. In contrast, glutathione-S-transferase activity remained constant despite a decline in the intracellular reduced glutathione content. 3. Epoxide hydrolase activity altered throughout time in culture, with an initial decrease in activity followed by a marked increase between days 7 and 10 after passage. 4. These results indicate the importance of standardizing the protocol with regard to the timing of experiments within the growth period of the cells when using hepatoma cell lines for assessing the metabolism and cytotoxicity of chemicals.

The role of reduced glutathione and glutathione reductase in the cytotoxicity of chromium (VI) in osteoblasts.

It is accepted that to exert cytotoxicity and carcinogenicity chromium VI has to be reduced inside cells. The role of reduced glutathione (GSH) and glutathione reductase in the intracellular reduction of Cr VI was investigated using an immortalized rat osteoblast cell line, FFC. Alkaline phosphatase activity was the index of cytotoxicity measured. To investigate the role of GSH in Cr VI toxicity, GSH levels in the cells were elevated by pretreatment with L-cysteine, and depleted using buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. Intracellular GSH levels were not depleted during the metabolism of Cr VI. Depletion of GSH by BSO caused the cells to be more resistant to the toxicity of Cr VI, indicating that GSH is involved in reduction of the Cr VI. Inhibition of glutathione reductase by carmustine (BCNU) partially protected against the cytotoxicity of Cr VI irrespective of the intracellular GSH. The cytotoxic response was similar if cells were pretreated with BCNU plus L-cysteine, or with BCNU plus BSO, although the GSH levels were markedly different. The results indicate that glutathione reductase plays an important role in the intracellular reduction of Cr VI in osteoblasts.

Measurement of the intracellular distribution of reduced glutathione in cultured rat hepatocytes using monochlorobimane and confocal laser scanning microscopy

Intracellular reduced glutathione (GSH) plays a key role in protecting cells from toxicity by maintaining intracellular redox status, conjugating with electrophilic xenobiotics and free radicals, and detoxifying reactive peroxides. Several toxic chemicals interact with GSH during their metabolism, and in many cases it would be advantageous to monitor intracellular GSH distribution during that process. We present a novel method to monitor intracellular GSH levels utilising a new laser light source, InGaN laser, for confocal microscopy and fluorescent detection of monochlorobimane (mBCl) binding to GSH. The sensitivity of the method was compared with that obtained using o-phthalaldehyde (OPT) as a fluorochrome. In the presence of a source of glutathione S-transferase (GST), mBCl was specific for GSH, forming a fluorescent conjugate that was retained in hepatocytes for at least 35 min. mBCl was able to detect the GSH depleting effects caused by progressive inhibition of GSH synthesis by increasing concentrations of buthionine sulfoximine. It effectively monitored the rapid effects of menadione and chromium VI metabolism on intracellular GSH levels in the cytosol and nuclear compartments of the cells. The combination of a specific stain, a novel laser light source and confocal microscopy provide a valuable system for mechanistic studies of intracellular GSH distribution in toxicology studies.

The influence of crosslinking agents and diamines on the pore size, morphology and the biological stability of collagen sponges and their effect on cell penetration through the sponge matrix

Artificial skin substitutes based on autologous keratinocytes cultured on collagen substrata are being developed for treating patients with severe burns. The properties of the collagen substrate can be manipulated, for example, by crosslinking, to optimize desirable properties such as cell growth and penetration into the substrate, biological stability and mechanical strength. Collagen sponges crosslinked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) and the diamine, diaminohexane, were used to determine the effect of crosslinking on pore size and morphology, on the stability of the crosslinked sponges both in cell culture media and during incubation with collagenase, and on the penetration of keratinocytes and fibroblasts through the sponge matrix. Crosslinking of the sponges reduced the pore size, particularly at the surface, and altered sponge morphology. After crosslinking the collagen fibers were thinner, and appeared lacy and delicate. Crosslinking also influenced sponge stability. In keratinocyte serum-free medium the pore size of plain collagen sponges increased with increasing incubation time, and crosslinking appeared to prevent this, and may have stabilized sponge structure. Incubation in serum-containing Dulbecco’s minimum essential medium caused a marked reduction in pore size in both plain collagen and crosslinked collagen sponges. Crosslinking did not appear to influence this cell-free contraction of collagen sponges. Treatment of sponges with EDAC markedly increased the resistance of sponges to collagenase digestion. The penetration of both keratinocytes and fibroblasts was retarded by crosslinking the sponges. Fibroblasts penetrated through the sponges to a greater extent than keratinocytes, and their proliferation rate was faster. The total number of cells populating the crosslinked sponges after 10 days culture was approximately 50% of that on untreated collagen sponges. The mechanism responsible for this effect was different with the two crosslinkers used. Diaminohexane appeared to inhibit cell growth, whereas EDAC may have caused a decrease in cell adhesion to the sponges, without an apparent inhibition of growth rate. In terms of morphology, fibroblasts were elongated to a greater extent on crosslinked sponges, and alligned themselves along the collagen fibers. Keratinocytes grew in colonies on untreated sponges, but on crosslinked sponges they grew in isolation, with minimal cell–cell interactions. It may be necessary to reach a compromise to obtain the best combination of properties for using collagen sponges as substrata for artificial skin substitutes.

The role of glutathione reductase in the cytotoxicity of chromium (VI) in isolated rat hepatocytes

Chromium (VI) is an environmental and occupational carcinogen, and it is accepted that intracellular reduction is necessary for DNA damage and cytotoxicity. We have investigated the interaction of Cr(VI) with hepatocytes in vitro to determine the contribution of various hepatic enzymes to the reduction of Cr(VI). Cr(VI) caused a dose-dependent decrease in cell viability and intracellular reduced glutathione (GSH) levels between 100 and 500 microM within 3 h exposure of hepatocytes. Both DT-diaphorase and cytochrome P450 play only a minor role in detoxifying Cr(VI) and/or its metabolites. (GSH) appears to act as a non-enzymatic reductant, reducing Cr(VI) to a toxic form. The evidence for this is two-fold. Firstly, GSH was depleted during the metabolism of Cr(VI) and, secondly, pretreatment of the cells with diethylmaleate to deplete GSH levels, partially protected the cells from Cr(VI) toxicity. Glutathione reductase appears to play an important role in the enzymatic reduction of Cr(VI) as inhibition of this enzyme by carmustine (BCNU) markedly protected the cells from cytotoxicity.

Novel quantitative methods for the determination of biomaterial cytotoxicity.

Two novel methods for the determination of biomaterial cytotoxicity using cell culture are presented. The methods combine a standardized protocol for producing extracts from medical devices with either the established MTT assay or a new fluorimetric assay. The suitability of both methods for evaluating the toxicity of candidate materials was demonstrated by resolution of the differences in the toxic effects of serial dilutions of a PVC extract on BHK21 and HT1080 cells. The tests yield highly reproducible, quantitative results and can be applied to materials in the usual physical forms applicable to artificial organs.