An investigation into the biological effects of indirect potable reuse water using zebrafish embryos.

Advanced treatment technologies are being assessed as a proactive measure to assist with the transformation of treated wastewater into a source of water for potable water production. We investigated the biological effects along an advanced water treatment pilot plant, using zebrafish embryos throughout early development. The study compared phenotypic observations with global transcriptome responses, enabling us to keep an open mind about the chemicals that might influence the biological activity. There was no evidence of acute toxicity at any treatment stage, but skeletal, cardiovascular and pigmentation changes occurred in a small proportion of embryos along the treatment process, and in a tap water; not detected in the aquarium water control. Reverse osmosis (RO) reduced the concentration of measured chemical contaminants in the water the most, while eliminating the occurrence of abnormalities detected in fish embryos. Conversely, advanced oxidation reversed the benefits of RO treatment by increasing the frequency of teratogenic and sub-lethal abnormalities seen. Using the molecular responses of zebrafish embryos to different IPR water, we report the bioactivity within the water at different stages of advanced treatment and associate these to perturbed biological functions. Transcriptomic analysis revealed alterations to the retinoid system, which was consistent with the observed teratogenic effects. Changes to tryptophan metabolism (associated with the production of melatonin required for the control of normal circadian rhythms) and somatolactin-beta (associated with normal pigmentation in fish) were also found. We show that underexplored forms of biological activity occur in treated wastewater effluent, and/or may be created depending on the type of advanced treatment process used. By integrating the available analytical chemistry we highlight chemical groups associated to this response. Our study shows that more detailed and in-depth characterisation of chemicals and biological pathways associated with advanced treatment water systems are needed to mitigate possible risks to downstream organisms.

Efficacy, safety and feasibility of intravenous iloprost in the domiciliary treatment of patients with ischemic disease of the lower limbs.

OBJECTIVE: Intravenous iloprost is an important option in the treatment of ischemic disease of the lower limbs; however, the administration of therapy is frequently compromised because of the need for long cycles of infusion in a hospital setting. The aim of the study is to evaluate the efficacy, safety, feasibility, and the economic impact of infusion therapy in the outpatient setting. PATIENTS AND METHODS: Twenty-four consecutive patients were treated with iloprost at their homes where they were administered a slow rate of infusion for 24 hours a day, during 9.9 ± 2.3 days, with a portable syringe pump (Infonde®). RESULTS: The clinical condition of patients evaluated with the modified SVS/ISCVS scale significantly improved after treatment (+1.29 ± 1.04 points vs. baseline, p<0.001). The drug was well tolerated; neither significant adverse events associated with medication nor problems related to venous access were recorded at home. Ninety-six percent of patients successfully completed the entire treatment cycle, and the evaluation questionnaire showed a high acceptance of the therapy. From the perspective of the hospital authority, lower direct medical costs were estimated for the domiciliary infusion process compared with the inpatient infusion setting. CONCLUSIONS: Treatment with iloprost in the outpatient setting is effective, safe, feasible, and more acceptable to patients than infusion at the hospital. In addition, it has a favorable economic and organizational impact on the medical ward.

Effect of a wound cleansing solution on wound bed preparation and inflammation in chronic wounds: a single-blind RCT.

OBJECTIVE: Research into surfactant solutions for the debridement of chronic wounds suggests that surfactants may support wound bed preparation (WBP) in chronic wounds, however their efficacy has not been evaluated in randomised controlled trials (RCTs). Our aim was to assess the clinical efficacy of a propylbetaine-polihexanide (PP) solution versus normal saline (NS) solution in WBP, assessing inflammatory signs and wound size reduction in patients with pressure ulcers (PUs) or vascular leg ulcers. METHOD: In a single-blinded randomised controlled trial (RCT) patients were randomly allocated to two groups and treated with either propylbetaine-polihexanide (PP) solution (Prontosan) or NS. Wounds were assessed using the Bates-Jensen wound assessment tool (BWAT). Assessments took place at inclusion (T0), day 7 (T1), day 14 (T2), day 21 (T3), and day 28 (T4). Outcomes were analysed using a two-tailed Student's t-test. RESULTS: A total of 289 patients were included. Both groups had similar demographics, clinical status, and wound characteristics. Data analysis showed statistically significant differences between T0 and T4 for the following outcomes: BWAT total score, p=0.0248; BWAT score for inflammatory items, p=0.03; BWAT scores for wound size reduction (p=0.049) and granulation tissue improvement (p=0.043), all in favour of PP. The assessment of pain did not show any significant difference between the two groups. CONCLUSION: The study results showed significantly higher efficacy of the PP solution versus NS solution, in reducing inflammatory signs and accelerating the healing of vascular leg ulcers and PUs. This evidence supports the update of protocols for the care of chronic wounds. DECLARATION OF INTEREST: The authors have no conflict of interest regarding this research. This is an investigator initiated trial. B. Braun Milano SpA kindly provided the material under investigation for both treatment groups, and paid the Ethics Committees' application fees in all participating centres.

Eukaryotic translation initiation factor 6 is a novel regulator of reactive oxygen species-dependent megakaryocyte maturation.

BACKGROUND: Ribosomopathies constitute a class of inherited disorders characterized by defects in ribosome biogenesis and function. Classically, bone marrow (BM) failure is a clinical symptom shared between these syndromes, including Shwachman-Bodian-Diamond syndrome (SBDS). Eukaryotic translation initiation factor 6 (eIF6) is a critical translation factor that rescues the quasilethal effect of the loss of the SBDS protein. OBJECTIVES: To determine whether eIF6 activity is necessary for BM development. METHODS: We used eIF6(+/-) mice and primary BM megakaryocytes to investigate the involvement of eIF6 in the regulation of hematopoiesis. RESULTS: We provide evidence that reduced eIF6 expression negatively impacts on megakaryopoiesis. We show that inhibition of eIF6 leads to a reduction in cell size and mean ploidy level of megakaryocytes and a delay in megakaryocyte maturation by blocking the G1 /S transition. Consistent with this phenotype, only few megakaryocyte-forming proplatelets were found in eIF6(+/-) cells. We also discovered that, in eIF6(+/-) cells, the steady-state abundance of mitochondrial respiratory chain complex I-encoding mRNAs is decreased, resulting in decreased reactive oxygen species (ROS) production. Intriguingly, connectivity map analysis showed that eIF6-mediated changes overlap with specific translational inhibitors. eIF6 is a translation factor acting downstream of insulin/phorbol 12-myristate 13-acetate (PMA) stimulation. PMA treatment significantly restored eIF6(+/-) megakaryocyte maturation, indicating that activation of eIF6 is essential for the rescue of the phenotype. CONCLUSIONS: Taken together, our results show a role for eIF6-driven translation in megakaryocyte development, and unveil the novel connection between translational control and ROS production in this cell subset.

Identification and angiogenic role of the novel tumor endothelial marker CLEC14A.

Tumor endothelial markers (TEMs) that are highly expressed in human tumor vasculature compared with vasculature in normal tissue hold clear therapeutic potential. We report that the C-type lectin CLEC14A is a novel TEM. Immunohistochemical and immunofluorescence staining of tissue arrays has shown that CLEC14A is strongly expressed in tumor vasculature when compared with vessels in normal tissue. CLEC14A overexpression in tumor vessels was seen in a wide range of solid tumor types. Functional studies showed that CLEC14A induces filopodia and facilitates endothelial migration, tube formation and vascular development in zebrafish that is, CLEC14A regulates pro-angiogenic phenotypes. CLEC14A antisera inhibited cell migration and tube formation, suggesting that anti-CLEC14A antibodies may have anti-angiogenic activity. Finally, in endothelial cultures, expression of CLEC14A increased at low shear stress, and we hypothesize that low shear stress due to poor blood flow in the disorganized tumor vasculature induces expression of CLEC14A on tumor vessels and pro-angiogenic phenotypes.

Hepatic transcriptomic profiles of European flounder (Platichthys flesus) from field sites and computational approaches to predict site from stress gene responses following exposure to model toxicants.

Genomic technologies offer opportunities to gain a more global assessment of the health status of an organism through an understanding of the functional pathways that are responding to pollutant exposure. We have developed a 13,000 clone cDNA toxicogenomics microarray for Platichthys flesus, the European flounder (EU-GENIPOL Project). We aimed to distinguish the origins of flounder taken from six sampling sites of different pollution status in Northern Europe according to their hepatic gene expression profile using bioinformatic approaches. To determine which gene expression differences may relate to pollutant impact, we have completed complementary laboratory exposures of flounder to selected toxicants and determined the associated gene expression profiles. Using multivariate variable selection coupled with a statistical modelling procedure (GALGO) we can predict geographical site but the accuracy is limited to specific sites. The search space for a combination of genes that effectively predicts class membership is very large, however, by combining the signatures derived from acute laboratory exposure to individual chemicals to limit the search space, a very accurate model for classification of all the different environmental sites was achieved. The final model utilised the expression profiles of 16 clones and validation with a qPCR array comprising these genes correctly assigned the site of origin for fish obtained from three of the sites in an independent sampling. These data would imply that the gene expression fingerprints obtained with these arrays are primarily attributable to variations in chemical pollutant responses at the different sites, indicating their potential utility in environmental impact assessment.

Expression analysis of immune response genes of Müller cells infected with Toxoplasma gondii.

PURPOSE: To investigate changes in immune response genes following Toxoplasma gondii infection of Müller cells. METHODS: Human Müller cells were infected or mock infected with two strains of T. gondii (RH and Prugniaud). RNA and supernatants were collected from infected and uninfected cells at 2 and 24 h. RNA from the two time points were compared using a custom made DNA microarray. Real time PCR or human cytokine antibody array was used to confirm up-regulation of immune molecules. RESULTS: Gene expression in infected cells showed up-regulation of CCL2, IL-6, CXCL8, and CXCL2. CCL2 and CXCL2 gene expression was confirmed by real time PCR. IL-6 and CXCL8 protein production was confirmed by a cytokine antibody array. IL-4 production was observed by cytokine antibody array but not by DNA microarray. In contrast, infection with T. gondii did not induce interferon-gamma (IFNgamma) and IL-12 expression, molecules conventionally associated with the inter-conversion of tachyzoite to bradyzoite. CONCLUSION: These results indicate that while in vitro infected Müller cells may be capable of inducing an immune response by attracting blood-borne leucocytes, they do not appear able to directly control the proliferation of T. gondii.

Identification of differentially expressed genes in coronary atherosclerotic plaques from patients with stable or unstable angina by cDNA array analysis.

The composition of atherosclerotic plaques is a crucial factor in determining rupture, thrombosis and clinical events. In this study, we analyzed gene expression in coronary plaques from patients with stable or unstable angina using gene arrays. Total RNA was extracted from eight plaques collected by therapeutic directional coronary atherectomy. cDNA probes, generated by amplification, were hybridized to nylon arrays containing 482 genes. Here we report the results for the inflammation, adhesion and hemostasis subsets. Many genes not previously associated with atherosclerosis, such as the lymphocyte adhesion molecule MadCAM, were expressed in the plaques. anova analysis showed higher tissue factor (TF) expression in unstable angina samples. Five genes were expressed at lower levels in unstable angina samples: anticoagulant protein S, cyclooxygenase (COX)-1, interleukin (IL)-7 and chemokines monocyte chemotactic protein (MCP)-1 and -2. Gene arrays provide a new approach to study plaque composition and identify candidate markers of plaque instability.

Methods and approaches in the analysis of gene expression data.

The application of high-density DNA array technology to monitor gene transcription has been responsible for a real paradigm shift in biology. The majority of research groups now have the ability to measure the expression of a significant proportion of the human genome in a single experiment, resulting in an unprecedented volume of data being made available to the scientific community. As a consequence of this, the storage, analysis and interpretation of this information present a major challenge. In the field of immunology the analysis of gene expression profiles has opened new areas of investigation. The study of cellular responses has revealed that cells respond to an activation signal with waves of co-ordinated gene expression profiles and that the components of these responses are the key to understanding the specific mechanisms which lead to phenotypic differentiation. The discovery of 'cell type specific' gene expression signatures have also helped the interpretation of the mechanisms leading to disease progression. Here we review the principles behind the most commonly used data analysis methods and discuss the approaches that have been employed in immunological research.

The comparison of gene expression from multiple cDNA libraries.

We describe a method for comparing the abundance of gene transcripts in cDNA libraries. This method allows for the comparison of gene expression in any number of libraries, in a single statistical analysis, to identify differentially expressed genes. Such genes may be of potential biological or pharmaceutical relevance. The formula that we derive is essentially the entropy of a partitioning of genes among cDNA libraries. This work goes beyond previously published analyses, which can either compare only two libraries, or identify a single outlier in a group of libraries. This work also addresses the problem of false positives associated with repeating the test on many thousands of genes. A randomization procedure is described that provides a quantitative measure of the degree of belief in the results; the results are further verified by considering a theoretically derived large deviations rate for the test statistic. As an example, the analysis is applied to four prostate cancer libraries from the Cancer Genome Anatomy Project. The analysis identifies biologically relevant genes that are differentially expressed in the different tumor cell types.

Maternal expression and early zygotic regulation of the Hox3/zen gene in the grasshopper Schistocerca gregaria.

In insects, a key step in the early patterning of the egg is to distinguish the primordium of the embryo proper from those regions that will form extra-embryonic membranes. In Drosophila, where these processes are well understood, the structure of the extra-embryonic membranes is highly derived. The distinct amnion and serosa typical of lower insects is replaced by a single, fused, and much reduced membrane, the amnioserosa, which never secretes an embryonic cuticle. We have used the Zen gene as a marker to study the formation of the extra-embryonic membranes, and other aspects of early embryonic patterning, in the grasshopper Schistocerca gregaria (African Plague Locust). Zen genes are derived from Hox genes, but in Drosophila they appear to have lost any role in patterning the A/P axis of the embryo; instead, they are involved in D/V patterning and the specification of the extra-embryonic membranes. We show that the Schistocerca zen gene is expressed during embryogenesis in three distinct phases. The first of these is during cleavage, when Sgzen is transiently expressed in all energids that reach the cell surface. The second phase of expression initiates in a ring of "necklace cells" that surround the forming embryo, and demarcate the boundary between the amnion and serosa. This leads to expression throughout the serosa. The final phase of expression is in the amnion, after this has separated from the serosa. This complex pattern implies that the role of Sgzen in Schistocerca is not limited solely to the specification of cell identity in the extra-embryonic membranes. We also report that the Schistocerca zen gene is expressed maternally, unlike its Drosophila and Tribolium counterparts. A distinct maternal transcript, and maternal Zen protein, accumulate in the developing oocyte from early post-meiotic stages. They remain uniformly distributed in the oocyte cytoplasm until late vitellogenic stages, when the protein and RNA become somewhat concentrated at the egg cortex and in the posterior polar cap of the oocyte, probably by passive exclusion from the yolk. The cytoplasmic localization of Sgzen protein in the oocyte, and at some stages during embryogenesis, implies that nuclear exclusion of this transcription factor is specifically controlled.

Class 3 Hox genes in insects and the origin of zen.

We have cloned, from a beetle and a locust, genes that are homologous to the class 3 Hox genes of vertebrates. Outside the homeobox they share sequence motifs with the Drosophila zerknüllt (zen) and z2 genes, and like zen, are expressed only in extraembryonic membranes. We conclude that the zen genes of Drosophila derive from a Hox class 3 sequence that formed part of the common ancestral Hox cluster, but that in insects this (Hox) gene has lost its role in patterning the anterio-posterior axis of the embryo, and acquired a new function. In the lineage leading to Drosophila, the zen genes have diverged particularly rapidly.

Dax, a locust Hox gene related to fushi-tarazu but showing no pair-rule expression.

We describe an unusual Antennapedia class homeobox gene from the grasshopper Schistocerca gregaria (Orthoptera, African Plague Locust). Its sequence is not sufficiently similar to that of any other insect Hom-Hox gene to identify it unambiguously, but short conserved elements suggest a relationship to the segmentation gene fushi-tarazu, (ftz). We term it Sg Dax (divergent Antennapedia class homeobox gene). Antibodies raised against the protein encoded by this gene reveal two phases of expression during embryogenesis. In the early embryo, it is a marker for the posterior part of the forming embryonic primordium, and subsequently for the posterior part of the growing germ band. In older embryos, it labels a subset of neural precursor cells in each trunk segment, very similar to that defined by the expression of fushi tarazu (ftz) in Drosophila. We suggest that Schistocerca Dax and Drosophila ftz are homologous members of a gene family whose members are diverging relatively rapidly, both in terms of sequence and role in early development.

Molybdenum(VI) salts convert the xanthine oxidoreductase apoprotein into the active enzyme in mouse L929 fibroblastic cells.

The mouse L929 fibroblastic cell line presents low, but detectable, levels of the mRNA encoding xanthine oxidoreductase under basal conditions, and it responds to type I and type II interferons by inducing the expression of the transcript [Falciani, Ghezzi, Terao, Cazzaniga, and Garattini (1992) Biochem. J. 285, 1001-1008]. This cell line, however, does not show any detectable amount of xanthine oxidoreductase enzymic activity, either before or after treatment with the cytokines. Molybdenum(VI) salts, in the millimolar range, are capable of activating xanthine oxidoreductase in L929 cells both under basal conditions and after treatment with interferon-alpha. The increase is observed in mouse L929 as well as in clones derived from it, but not in many other human and mouse cell lines. The induction observed in L929 cells is post-translational in nature and it is insensitive to cycloheximide, indicating that the molybdenum ion converts a pool of inactive xanthine oxidoreductase apoenzyme into its holoenzymic form. When grown in the absence of sodium molybdate, the L929 cell line has undetectable intracellular levels of the molybdenum cofactor, since the cell extracts are unable to complement the nitrate reductase defect of the nit-1 mutant of Neurospora crassa. L929 cells grown in the presence of millimolar concentrations of sodium molybdate, however, become competent to complement the nit-1 defect. L929 cells accumulate molybdenum ion inside the intracellular compartment as efficiently as TEnd cells, a mouse endothelial cell line that expresses xanthine oxidoreductase activity both under basal conditions and after treatment with interferon-gamma, suggesting that L929 cells have a defect in one or more of the metabolic steps leading to the synthesis of the molybdenum cofactor.

The evolving role of Hox genes in arthropods.

Comparisons between Hox genes in different arthropods suggest that the diversity of Antennapedia-class homeotic genes present in modern insects had already arisen before the divergence of insects and crustaceans, probably during the Cambrian. Hox gene duplications are therefore unlikely to have occurred concomitantly with trunk segment diversification in the lineage leading to insects. Available data suggest that domains of homeotic gene expression are also generally conserved among insects, but changes in Hox gene regulation may have played a significant role in segment diversification. Differences that have been documented alter specific aspects of Hox gene regulation within segments and correlate with alterations in segment morphology rather than overt homeotic transformations. The Drosophila Hox cluster contains several homeobox genes that are not homeotic genes--bicoid, fushi-tarazu and zen. the role of these genes during early development has been studied in some detail. It appears to be without parallel among the vertebrate Hox genes. No well conserved homologues of these genes have been found in other taxa, suggesting that they are evolving faster than the homeotic genes. Relatively divergent Antp-class genes isolated from other insects are probably homologues of fushi-tarazu, but these are almost unrecognisable outside of their homeodomains, and have accumulated approximately 10 times as many changes in their homeodomains as have homeotic genes in the same comparisons. They show conserved patterns of expression in the nervous system, but not during early development.

Cytokine induction of haem oxygenase mRNA in mouse liver. Interleukin 1 transcriptionally activates the haem oxygenase gene.

Accumulation of the mRNA coding for haem oxygenase (HO, EC 1.14.99.3) was stimulated by treating mice with endotoxin (lipopolysaccharide, LPS; 20 micrograms/mouse intraperitoneally), suggesting that haem catabolism is a target of infection and inflammation in vivo. Therefore various cytokines, possible mediators for the biological responses to LPS, were administered intraperitoneally to mice, and the levels of HO mRNA were measured by Northern-blotting analysis using the rat HO cDNA as a probe [Shibahara, Müller, Taguchi and Yoshida (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7865-7869]. Marked induction of HO mRNA was observed 2 h after administration of interleukin 1 (IL-1) (34-fold) and tumour necrosis factor (19.5-fold) (5 micrograms/mouse), whereas interleukin 6 (6.2 micrograms/mouse) was much less active (3.5-fold) and interleukin 2 (25 micrograms/mouse) and interferon-gamma (3 micrograms/mouse) were ineffective. HO mRNA induced by the cytokines of LPS accumulated rapidly (maximum at 1-2 h after administration), preceding the elevation of HO enzymic activity. Treatment of mice with IL-1 stimulated the transcription of the HO gene by 4-fold, as assessed by in vitro nuclear-run-on assay. These results indicate that enzymic haem catabolism in the liver is a process inducible in vivo by inflammatory cytokines, which up-regulate HO synthesis at the transcriptional level. Increased removal of haem might be part of the protective mechanisms elicited by the acute-phase response, possibly to reduce the pro-oxidant state of the cell.

Regulation of the 202 gene expression by interferons in L929 cells.

Type I and II interferons (IFNs) stimulate the expression of the 202 and 2'-5' oligoadenylate synthetase (OASE) genes in L929, NIH 3T3 and LM-TK- fibroblastic cell lines. In two other cell lines, B16 melanoma and F9 teratocarcinoma, these cytokines induce OASE but not the 202 mRNA. In L929 cells, IFN-alpha induces the 202 mRNA at concentrations between 10 and 10(3) units/ml. To achieve maximal induction of the 202 mRNA, continuous exposure of L929 cells to IFN-alpha is necessary, whereas 30 minutes of exposure are sufficient to trigger maximal upregulation of the OASE transcript. The induction of the 202 mRNA is the consequence of both transcriptional and post-transcriptional events. Cycloheximide, a known inhibitor of protein synthesis, does not block the induction of 202 mRNA by IFN-alpha, demonstrating that new protein synthesis is not required for this effect. Protein kinase C, arachidonic acid metabolism via the cyclooxygenase or the lipoxygenase pathways and cAMP are not involved as second messengers in the induction of the 202 mRNA by IFN-alpha in L929 cells.

Interferons induce xanthine dehydrogenase gene expression in L929 cells.

Human interferon-alpha A/D (Bg/II) (IFN-alpha A/D) and mouse interferon-gamma (IFN-gamma) are shown to induce xanthine dehydrogenase (XD) mRNA in L929 fibroblastic cells. XD mRNA accumulation after IFN-alpha A/D treatment is relatively fast, being already evident after 4 h and reaching its maximum after 24 h. IFN-alpha A/D is active in inducing XD mRNA at 0.1 unit/ml and it is maximally active at 10(3) units/ml. The half-life of the XD message is unaffected by IFN-alpha A/D treatment, whereas the transcriptional activity of the XD gene and the concentrations of XD heterogeneous nuclear RNA are increased by 2- and 6-fold respectively. The effect of IFN-alpha A/D on XD mRNA is insensitive to cycloheximide, suggesting that protein synthesis de novo is not required. Experiments conducted with specific inhibitors suggest that protein kinase C, cyclic AMP and arachidonic acid metabolites derived from lipoxygenase or cyclooxygenase do not act as second-messenger molecules in the induction of XD mRNA by IFN-alpha A/D. XD mRNA is also induced in NIH3T3 fibroblastic cells, but not in F9 teratocarcinoma or B16 melanoma cells after treatment with IFN-alpha A/D. NIH3T3 are the only cells so far tested that have detectable XD and xanthine oxidase activities under basal conditions and after IFN-alpha A/D treatment, although their responsiveness to the cytokine is much less than that observed in L929 cells.

Molecular cloning of a cDNA coding for mouse liver xanthine dehydrogenase. Regulation of its transcript by interferons in vivo.

The cDNA coding for xanthine dehydrogenase (XD) is isolated from mouse liver mRNA by cross-hybridization with a DNA fragment of the Drosophila melanogaster homologue. Two lambda bacteriophage overlapping clones represent the copy of a 4538-nucleotide-residue-long transcript with an open reading frame of 4005 nucleotide residues, coding for a putative polypeptide of 1335 amino acid residues. Comparison of the deduced amino acid sequence of the mouse XD with those of the Drosophila and the rat homologues shows a high conservation of this protein (55% identity between mouse and Drosophila, and 94% identity between mouse and rat). RNA blotting analysis demonstrates that interferon-alpha (IFN-alpha) and its inducers, i.e. poly(I).poly(C), bacterial lipopolysaccharide (LPS) and tilorone (2,7-bis-[2-(diethylamino)ethoxy]fluoren-9-one), increase the expression of XD mRNA in liver. Poly(I).poly(C) also induces XD mRNA in several other tissues in vivo. Protein synthesis de novo is not required for the elevation of XD mRNA after IFN-alpha treatment, since cycloheximide does not block the induction. The elevation of XD mRNA concentration is relatively fast and precedes the induction of both XD and xanthine oxidase (XO) enzymic activities.