Gabrielle M. Hawksworth

Induction, Regulation, Degradation, and Biological Significance of Mammalian Metallothioneins

MTs are small cysteine-rich metal-binding proteins found in many species and, although there are differences between them, it is of note that they have a great deal of sequence and structural homology. Mammalian MTs are 61 or 62 amino acid polypep-tides containing 20 conserved cysteine residues that underpin the binding of metals. The existence of MT across species is indicative of its biological demand, while the conservation of cysteines indicates that these are undoubtedly central to the function of this protein. Four MT isoforms have been found so far, MT-1, MT-2, MT-3, and MT-4, but these also have subtypes with 17 MT genes identified in man, of which 10 are known to be functional. Different cells express different MT isoforms with varying levels of expression perhaps as a result of the different function of each isoform. Even different metals induce and bind to MTs to different extents. Over 40 years of research into MT have yielded much information on this protein, but have failed to assign to it a definitive biological role. The fact that multiple MT isoforms exist, and the great variety of substances and agents that act as inducers, further complicates the search for the biological role of MTs. This article reviews the current knowledge on the biochemistry, induction, regulation, and degradation of this protein in mammals, with a particular emphasis on human MTs. It also considers the possible biological roles of this protein, which include participation in cell proliferation and apoptosis, homeostasis of essential metals, cellular free radical scavenging, and metal detoxification.

Human adult hepatocytes in primary monolayer culture: Maintenance of mixed function oxidase and conjugation pathways of drug metabolism

The stabilities of several drug oxidation and conjugation pathways in human adult hepatocytes have been investigated during 72 hr culture. Cytochrome P-450-dependent mixed function oxidase was measured by the O-dealkylations of ethoxyresorufin (EROD), pentoxyresorufin (PROD) and benzyloxyresorufin (BROD), which are probes for different isozymes of cytochrome P-450 in the rat. EROD declined to 64% of initial fresh cell values after 72 hr in culture, whereas PROD increased to 162% and BROD remained relatively constant. Addition of phenobarbitone to the culture medium selectively increased PROD to a greater extent than EROD and did not affect BROD. NADPH-cytochrome c reductase and NADH-cytochrome b5 reductase were markedly labile during culture, declining to 32% and 22% of fresh cell values respectively. Epoxide hydrolase (EH) showed a large transient increase (2-5-fold) in enzyme activity 24 hr after culture, declining to fresh cell values by 48 hr. UDP-glucuronyltransferase (GT) activity towards phenolphthalein and 1-naphthol also increased (2-3-fold) during the 72 hr of culture, the greater and more rapid increase being observed with phenolphthalein glucuronidation. Sulphotransferase activity declined rapidly within 24 hr of culture, whereas reduced glutathione (GSH) levels and GSH conjugation were maintained at fresh cell values for 72 hr.

Exposure of cultured human proximal tubular cells to cadmium, mercury, zinc and bismuth: toxicity and metallothionein induction

The kidney, in particular the proximal convoluted tubule, is a major target site for the toxic effects of various metals. However, little is known about the early effects of these metals after acute exposure in man. In the present study we have evaluated the toxicity of several inorganic metal compounds (CdCl2, HgCl2, ZnCl2, and Bi(NO3)3) and the induction of metallothionein by these compounds in cultured human proximal tubular (HPT) cells for up to 4 days. The results showed that bismuth was not toxic even at the highest dose (100 microM) used, while zinc, cadmium and mercury exhibited varying degrees of toxicity, zinc being the least toxic and mercury the most potent. A significant degree of interindividual variation between the different isolates used in these experiments was also observed. All metals used in the present study induced MT, as revealed by immunocytochemistry. All metals showed maximal induction between 1 and 3 days after treatment. Although a certain amount of constitutive MT was present in the cultures, the intensity of the staining varied with time in culture and between the different isolates studied. No correlation could be made between the intensity of the staining in control cultures (indicating total amount of constitutive MT) and the susceptibility of a given isolate to metal toxicity. Furthermore, no correlation could be made between metal-induced MT and the susceptibility of a given isolate to that particular metal.

Kinetics of paraquat elimination in the dog

Abstract The elimination and distribution kinetics of paraquat and the effect of paraquat-induced renal damage on these parameters have been studied in the dog. After intravenous administration, low doses (30–50 μg/kg) of paraquat were rapidly excreted in the urine at a clearance rate in excess of glomerular filtration rate, indicating that paraquat was eliminated by active secretion. This secretion could be inhibited by competition with N′-methylnicotinamide, a quaternary ammonium compound. Large doses (20 mg/kg) of paraquat induced renal failure, the extent of renal function impairment depending on the dose. The plasma concentration-time curve showed a triexponential decline, so that the elimination of paraquat could be described by a three-compartment open model. Simulation of paraquat levels in the peripheral compartments indicated that with early renal failure, fivefold higher levels would be achieved in the slow uptake compartment than under conditions of normal renal function. Since the energy-dependent accumulation of paraquat by the lung is a slow process, it was assumed that the slow uptake compartment included the lung. The implications of this are discussed in relation to the rational treatment of paraquat poisoning in man.

Cryopreservation of human adult hepatocytes for use in drug metabolism and toxicity studies

1. Human hepatocytes were cryopreserved for up to 14 days at -80 degrees C and the cryoprotection offered by different media investigated in terms of post-thaw cell viability and function. 2. Optimal cryoprotection was offered by a solution containing dimethylsulphoxide, propylene glycol, acetamide and polyethylene glycol 8000 in Leibowitz L15 medium. 3. The cytochrome P450 content and activities of the microsomal P450 dependent mixed function oxidase system were well maintained at above 70% of fresh cell values throughout the cryopreservation period. However, the activities of the cytosolic enzymes studied, glutathione S-transferase and glutathione reductase, were not well maintained; they declined to < 40% of fresh cell values after storage of cells for 14 days at -80 degrees C. The membrane environment may protect microsomal enzymes from denaturation by freeze-thaw damage. 4. After cryopreservation, viability of human hepatocytes was higher than that of rat hepatocytes preserved under identical conditions. For human cells maximum post-cryopreservation viability was 67% after 24 h at -80 degrees C; this declined to 49% after 14 days storage at -80 degrees C. In addition post-cryopreservation human hepatocytes remained > 70% viable when incubated at 37 degrees C in suspension compared with only 46% of rat hepatocytes. This indicates that human hepatocytes can withstand freeze-thaw damage better than those from rat. 5. The results of this study define optimal conditions for cryopreserving human hepatocytes. Although microsomal enzyme activities are retained post-cryopreservation, the decrease in viability of thawed cells upon incubation at 37 degrees C suggests that caution should be exercized when using cryopreserved cells to study integrated drug metabolizing pathways in man in vitro.

Mechanism of action of the antifibrogenic compound gliotoxin in rat liver cells

Gliotoxin has been shown to promote a reversal of liver fibrosis in an animal model of the disease although its mechanism of action in the liver is poorly defined. The effects of gliotoxin on activated hepatic stellate cells (HSCs) and hepatocytes have therefore been examined. Addition of gliotoxin (1.5 μM) to culture‐activated HSCs resulted in its rapid accumulation, resulting in increased levels of glutathione and apoptosis without any evidence of oxidative stress. In contrast, although hepatocytes also rapidly sequestered gliotoxin, cell death only occurred at high (50‐μM) concentrations of gliotoxin and by necrosis. At high concentrations, gliotoxin was metabolized by hepatocytes to a reduced (dithiol) metabolite and glutathione was rapidly oxidized. Fluorescent dye loading experiments showed that gliotoxin caused oxidative stress in hepatocytes. Antioxidants—but not thiol redox active compounds—inhibited both oxidative stress and necrosis in hepatocytes. In contrast, HSC apoptosis was not affected by antioxidants but was potently abrogated by thiol redox active compounds. The adenine nucleotide transporter (ANT) is implicated in mitochondrial‐dependent apoptosis. HSCs expressed predominantly nonliver ANT isoform 1, and gliotoxin treatment resulted in a thiol redox‐dependent alteration in ANT mobility in HSC extracts, but not hepatocyte extracts. In conclusion, these data suggest that gliotoxin stimulates the apoptosis of HSCs through a specific thiol redox‐dependent interaction with the ANT. Further understanding of this mechanism of cell death will aid in finding therapeutics that specifically stimulate HSC apoptosis in the liver, a promising approach to antifibrotic therapy. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2004;40:232–242.)

Cytokine-Stimulated Nitric Oxide Production in the Human Renal Proximal Tubule and Its Modulation by Natriuretic Peptides: A Novel Immunomodulatory Mechanism?

Although the importance of the human kidney in a variety of disease states is well recognised, the exact mechanisms involved remain unclear. Animal disease models suggest that while high local concentrations of nitric oxide (NO) may play a key role in the initiation and progression of renal disease, low levels may also be essential for normal renal function and cell protection, possibly explaining the variable reports of both beneficial and detrimental responses of renal disease models following NO inhibition. NO has both physiological and pathological roles and clearly a balance between these two primary roles is likely to prevail leading to the conclusion that partial rather than total inhibition of NO production may be beneficial. Despite increasing evidence for the role of NO from animal disease models, little is known of the role of NO and potential modulators within the human kidney. In this review we describe three series of studies during which we examined the ability of primary cultures of human proximal tubular cells to produce NO in response to inflammatory cytokines and the possible role of potential modulators such as the natriuretic peptides. Following challenge with the combination of inflammatory cytokines IL-1β, TNF-α, and IFN-γ, such cultures exhibit a time-dependent increase in inducible NO synthetase induction and corresponding NO production, an effect which was inhibited by L-NMMA. In the second series of studies we demonstrated that increasing concentrations of atrial natriuretic factor (ANF) or C(4–23)ANF could stimulate a time- and concentration-dependent increase in nitric oxide production which was again abolished by L-NMMA. These results suggested that ANF acting at the natriuretic peptide receptor C could stimulate nitric oxide production in human proximal tubular cells. In the final series of studies we demonstrated that pro-inflammatory cytokine-induced nitric oxide production could be inhibited by ANF, brain natriuretic peptide, C-type natriuretic peptide or C(4–23)ANF. The actions of the natriuretic peptides and C(4–23)ANF was to return pro-inflammatory nitric oxide production to those observed when human proximal tubular cells were incubated with ANF alone indicating that this inhibition was mediated via the natriuretic peptide receptor C. The function of NO in the kidney is unclear but undoubtedly it has both beneficial and detrimental actions which in health remain in balance. However, when the kidney is subjected to an immune challenge, high cytotoxic levels of NO are produced locally and appear to be responsible for local damage, unfortunately total inhibition of NO production during such disease states does not always result in benefit. Clearly total abolition of an NO response removes important integral protective actions such as vasodilation. In the ideal situation, treatment of disease processes related to NO excess would involve the inhibition of these high local levels while still protecting vital dependent mechanisms. We believe that the NO natriuretic peptide interaction, which we have reported in this review, places ANF in a unique position of being able to maintain the essential or protective actions of NO while inhibiting potentially cytotoxic or detrimental effects.

Lymphocyte metallothionein mRNA responds to marginal zinc intake in human volunteers.

Marginal Zn deficiency is thought to be prevalent in both developed and developing countries. However, the extent of Zn deficiency is not known, due to the lack of a reliable diagnostic indicator. Blood plasma and erythrocyte concentrations of metallothionein (MT) reflect Zn status, but measurement of MT is dependent on the availability of sensitive immunoassays. Our aim was to show whether measurement of T lymphocyte MT-2A mRNA, using a competitive reverse transcriptase (RT)--polymerase chain reaction (PCR) assay, could indicate Zn status in human subjects in a residential Zn-depletion study. In the study, the Zn intake of seven volunteers was maintained at 13.7 mg/d for 5 weeks (baseline) followed by 4.6 mg/d for 10 weeks (marginal intake) and then 13.7 mg/d (repletion) for 5 weeks. The quantitative assay was developed using standard techniques and concentrations of MT-2A mRNA were normalized by reference to beta-actin mRNA which was also measured by competitive RT--PCR assay. An alternative method of measuring the PCR product using capillary electrophoresis with laser-induced fluorescence detection was also evaluated. There was considerable inter-individual variation in MT-2A mRNA concentration and the mean level at the end of the baseline period was 10.3 (SE 3.7) fg MT-2A mRNA/pg beta-actin mRNA, which then decreased by 64 % during the low Zn intake period. After repletion, MT-2A mRNA returned to baseline concentrations. In contrast, plasma Zn was unchanged by marginal Zn intake or repletion. The effect of low Zn in all individuals was consistent. We conclude that this assay is a sensitive method of evaluating marginal changes in dietary Zn intake.

Generation of hepatocytes expressing functional cytochromes P450 from a pancreatic progenitor cell line in vitro

The proliferating AR42J-B13 pancreatic cell line is known to respond to glucocorticoid treatment by producing foci of cells that express the liver-specific albumin gene. We demonstrate that this cell line also expresses liver-specific or liver-enriched functional cytochrome P450 proteins when stimulated to trans-differentiate into hepatocytes by glucocorticoid. These data suggest that this cell line has an unusual ability to trans-differentiate into functional hepatocytes and that it could be possible to generate a limitless supply of functional hepatocyte-like cells in vitro.

Atrial natriuretic factor modulates nitric oxide production : an ANF-C receptor-mediated effect

Objectives: To investigate the possible immunomodulatory and regulatory functions of atrial natriuretic factor (ANF) and the natriuretic peptide C (NPR-C) receptor in the control of cytokine-stimulated nitric oxide production in primary cultures of human proximal tubular cells. Methods: Freshly prepared human proximal tubular cells were seeded on plastic plates and allowed to reach confluence. The confluent cells were then incubated with ANF or cycliC(4_23)ANF (c(4_23)ANF) alone, or preincubated with ANF or C(4_23)ANF before incubation with the nitric oxide-stimulating combination of cytokines interleukin-1|3 (10u/ml), tumour necrosis factor-cc (10ng/ml) and interferon-y (100 u/ml). Results: In the present series of experiments we have found that incubation of primary cultures of human proximal tubular cells with ANF or C(4_23)ANF stimulates nitric oxide production dose-dependently. Paradoxically, ANF acting via the NPR-C receptor also inhibits cytokine activation of the enzyme-inducible nitric oxide synthase via a cyclic GMP-independent mechanism. Both of these effects were reproduced by the NPR-C receptor-specific ligand cIn the present series of experiments we have found that incubatio4_23)ANF. Conclusions: These findings represent novel actions of ANF mediated via the NPR-C receptor. The results also provide a simple model system in which to study the subcellular mechanisms of NPR-C receptor activation.