Lead ingestion as a potential contributing factor to the decline in vulture populations in southern Africa.

Vulture populations in southern Africa have been on the decline for years, which unlike the Asian vulture crisis, has no one specific cause. Reasons attributable are deliberate and secondary poisonings, drowning, power line injuries, electrocutions, traditional medicine ("muti" trade) and calcium deficiencies. However, lead toxicity as a potential causal factor is hardly mentioned. The potential for lead toxicity needs to be considered as substantial game hunting occurs in the region with little regulatory control on bullet types. In this study, we determined the whole blood lead concentrations of captive and wild vulture populations in South Africa and Namibia (n=185). Results were compared to previous published ranges indicative of background exposure (<10µg/dL), non-toxic point exposure based upon the range established from captive birds and subclinical exposure. In general, whole blood lead concentrations were higher for wild African White-backed vultures (Gyps africanus)(AWBV) than Cape vultures (G. coprotheres)(CGV) at 15.54±12.63µg/dL vs 12.53±8.88µg/dL (non-significantly different), while in the Bearded vultures (Gypaetus barbatus) no indication of exposure was evident. Very similar exposures resulted irrespective of the birds being in captivity or under wild, free-roaming conditions. A proportion of wild birds did, however, appear to be exposed to another source of lead than purely environmental (±12% and 30.6% for AWBV and CGV respectively). One bird, which had a whole blood concentration of 100µg/dL, died soon after capture. To find the relationship between whole blood lead concentration and likely exposure factors, birds were compared by their rural/urban location, vicinity to mines and surrounding soil lead concentrations. With no relationship being present for the latter factors, we believe that this is evidence that the portion of southern African vultures being exposed to unknown source of lead, which we suggest arises from leaded ammunition remaining from hunting.

Toward a highly-detailed 3D pelvic model: approaching an ultra-specific level for surgical simulation and anatomical education.

The surgical anatomy of the pelvis is highly complex. Anorectal and urogenital dysfunctions occur frequently after pelvic oncological surgery and are mainly caused by surgical damage of the autonomic nerves. A highly-detailed 3D pelvic model could increase the anatomical knowledge and form a solid basis for a surgical simulation system. Currently, pelvic surgeons still rely on the preoperative interpretation of 2D diagnostic images. With a 3D simulation system, pelvic surgeons could simulate and train different scenes to enhance their preoperative knowledge and improve surgical outcome. To substantially enrich pelvic surgery and anatomical education, such a system must provide insight into the relation between the autonomic network, the lymphatic system, and endopelvic fasciae. Besides CT and MR images, Visible Human Datasets (VHDs) are widely used for 3D modeling, due to the high degree of anatomical detail represented in the cryosectional images. However, key surgical structures cannot be fully identified using VHDs and radiologic imaging techniques alone. Several unsolved anatomical problems must be elucidated as well. Therefore, adequate analysis on a microscopic level is inevitable. The development of a comprehensive anatomical atlas of the pelvis is no straightforward task. Such an endeavor involves several anatomical and technical challenges. This article surveys all existing 3D pelvic models, focusing on the level of anatomical detail. The use of VHDs in the 3D reconstruction of a highly-detailed pelvic model and the accompanying anatomical challenges will be discussed.

Cytotoxicity, morphological and ultrastructural effects induced by the neonicotinoid pesticide, imidacloprid, using a rat Leydig cell line (LC-540).

Imidacloprid is a systemic neonicotinoid insecticide widely used to combat agricultural pests and flea infestations in dogs and cats. Despite its low toxicity to mammals, imidacloprid is reported to cause male reproductive toxicity. This study evaluated the cytotoxic effects of 75-800 µM imidacloprid on a rat Leydig cell line (LC-540). The effect of exposure to 300, 400, and 500 µM imidacloprid on selected cytoskeletal proteins, mitochondrial morphology, lysosomal acidity, and ultrastructure were investigated. Cell viability was markedly reduced after 48 and 72 h of exposure to higher imidacloprid concentrations. The immunocytochemical analysis revealed that the cytoskeletal filaments exhibited disorganization, disruption, and perinuclear aggregation in treated LC-540 cells. Ultrastructurally, cytoplasmic vacuoles, autophagic vacuoles, lysosomes, and mitochondrial damage were detected. Changes in the mitochondrial morphology and lysosomes induced by imidacloprid were confirmed. The cytotoxicity of imidacloprid observed in LC-540 cells might be due to its mitochondrial damage and cytoskeletal protein disruption.

What is in a name? Scientific name changes of potentially poisonous plants and fungi in South Africa.

Changes over the past five decades in the scientific names of some potentially poisonous plants and toxigenic fungi in South Africa are briefly reviewed. Some of the reasons why taxonomists change names are highlighted. In recent years, DNA sequencing data have contributed considerably towards establishing phylogenetic relationships among plants, often resulting in changes in generic circumscription and, consequently, the names of species. Philosophical differences between the phylogenetic and the evolutionary schools of plant classification are briefly explained as these may manifest as different classifications for the same group of plants. Although choice of classification remains the prerogative of the end-user of plant names, in this review, the classifications for plants currently adopted by the South African National Biodiversity Institute (SANBI) in its online database, Plants of Southern Africa (POSA), were followed. Noteworthy generic changes include Pachystigma to Vangueria, Homeria to Moraea, and Urginia to Drimia. Following much controversy, the species native to southern Africa that were formerly treated as Acacia are now classified in either Vachellia or Senegalia, with the genus name Acacia being retained for the mainly Australian members of the group, the latter commonly known as wattles. Former southern African members of Acacia implicated in poisoning include Vachellia erioloba(camel thorn), Vachellia sieberiana var. woodii (paperbark thorn), and Senegalia caffra (common hook thorn).

The optimal management of the patient with COVID-19 pneumonia: HFNC, NIV/CPAP or mechanical ventilation?

The recent pandemic has seen unprecedented demand for respiratory support of patients with COVID-19 pneumonia, stretching services and clinicians. Yet despite the global numbers of patients treated, guidance is not clear on the correct choice of modality or the timing of escalation of therapy for an individual patient. This narrative review assesses the available literature on the best use of different modalities of respiratory support for an individual patient, and discusses benefits and risks of each, coupled with practical advice to improve outcomes. On current data, in an ideal context, it appears that as disease severity worsens, conventional oxygen therapy is not sufficient alone. In more severe disease, i.e. PaO2/FiO2 ratios below approximately 200, helmet-CPAP (continuous positive airway pressure) (although not widely available) may be superior to high-flow nasal cannula (HFNC) therapy or facemask non-invasive ventilation (NIV)/CPAP, and that facemask NIV/CPAP may be superior to HFNC, but with noted important complications, including risk of pneumothoraces. In an ideal context, invasive mechanical ventilation should not be delayed where indicated and available. Vitally, the choice of respiratory support should not be prescriptive but contextualised to each setting, as supply and demand of resources vary markedly between institutions. Over time, institutions should develop clear policies to guide clinicians before demand exceeds supply, and should frequently review best practice as evidence matures.

The pathology of acute Nolletia gariepina poisoning of cattle.

Toxicity in cattle by the shrub Nolletia gariepina was induced experimentally by intraruminal administration of 3 g/kg dried, milled plant material as a single dose. The animals had to be starved for 24 hours before dosing, as dosing on a full rumen did not induce any signs of toxicity during 5 days of observation and clinical pathology monitoring. Clinical signs were not specific and varied according to the duration (acute versus subacute) of the toxicological process. Clinical pathological parameters indicated renal and to a lesser extent hepatic damage, with raised serum concentrations of urea, creatinine, aspartate aminotransferase (AST) and gamma glutamyl transferase (GGT). Increased urinary sodium and potassium concentration and GGT activity, as well as proteinuria, were evident. Histological and electron microscopic examinations revealed acute renal tubular epithelial cell degeneration and necrosis, especially of the proximal convoluted tubules. Mild hepatocellular degeneration was also noticeable.

Solvent suppression techniques for coupling of size exclusion chromatography and 1H NMR using benchtop spectrometers at 43 and 62 MHz.

The characterisation of polymeric materials in their full complexity of chain length, monomeric composition, branching and functionalization is a tremendous challenge and is best tackled by tailored multi-dimensional coupled analytical and detection techniques. Herein, we focus on the improvement of an affordable but information rich 2D-method for polymer analysis: the online hyphenation of benchtop 1H NMR spectroscopy with size exclusion chromatography (SEC). The main benefit of this approach is correlated information of chain length (SEC) to chemical composition (1H NMR). Our setup combines SEC onflow with a benchtop NMR spectrometer at 43 or 62 MHz with chemical shift resolution as a robust detector. A detailed comparison of the two instruments is included considering, that only the 43 MHz instrument is equipped with a dedicated z-gradient enabling pulse sequences such as WET. The main challenge of this method is the very low concentration of species of interest after chromatographic separation. At typical SEC conditions, the analyte dilution is typically more than a factor of 1000:1 in a protonated solvent. Therefore, an efficient solvent signal suppression is needed. In this article, several suppression pulse sequences are explored like WET, WEFT, JNR and a simple one-pulse approach - some for the first time on this hardware. By choosing an optimal method, signal strength ratios of solvent to analyte of 1:1 or better are achievable on flow. To illustrate the broad range of possible applications, three typical cases of analyte to solvent signal proximity (no overlap, partial and full overlap) are discussed using typical polymers (PS, PMMA, PEMA) and solvents (chloroform and THF). For each case, several suppression methods are compared and evaluated using a set of numerical criteria (analyte signal suppression and broadening, solvent signal suppression, remaining solvent signal width).

Pharmacology of visceral pain: central factors.

Clinically, pain can be sub-classified into superficial, neuropathic and deep pain. Deep pain as a result of stimulation to structures such as the viscera is the most poorly understood and notoriously difficult to treat. The dorsal horn of the spinal cord is the gateway to conscious nociception and it is at this point in the pain processing pathway that the peripheral afferent input can be enhanced or inhibited by several mechanisms, the most important being central sensitisation. Long-term potentiation, another mechanism, can also be elicited in the spinal cord. Here nociceptor activity and/or peripheral tissue inflammation produces long-term changes in synaptic efficacy in the dorsal horns. This plays a major role in the generation of acute post-operative and post-traumatic pain, migraine and neuropathic pain. Behavioural consequences of central sensitisation can even be readily detected in human psychophysical experiments. Another important mechanism is 'wind-up', a form of homosynaptic activity-dependent plasticity characterised by a progressive increase in action potential output from dorsal horn neurones. There is an extensive body of literature which has highlighted the importance of central sensitisation. This review examines some of the most significant recent findings with regards to future pharmacology. As we are beginning to understand some of the mechanisms of central sensitisation and its importance in visceral pain, novel receptor sites have been identified, offering exciting possibilities with regards to future pharmacological development not only to visceral pain, but for pain management as a whole.

Zoonotic tuberculosis and brucellosis in Africa: neglected zoonoses or minor public-health issues? The outcomes of a multi-disciplinary workshop.

Late in 2007, veterinary, medical and anthropological professionals from Europe and Africa met in a 2-day workshop in Pretoria, South Africa, to evaluate the burden, surveillance and control of zoonotic tuberculosis and brucellosis in sub-Saharan Africa. Keynote presentations reviewed the burden of these diseases on human and livestock health, the existing diagnostic tools, and the available control methods. These presentations were followed by group discussions and the formulation of recommendations. The presence of Mycobacterium bovis and Brucella spp. in livestock was considered to be a serious threat to public health, since livestock and animal products are the only source of such infections in human beings. The impact of these pathogens on human health appears to be relatively marginal, however, when compared with Mycobacterium tuberculosis infections and drug resistance, HIV and malaria. Appropriate diagnostic tools are needed to improve the detection of M. bovis and Brucella spp. in humans. In livestock, the 'test-and-slaughter' approach and the pasteurization of milk, which have been used successfully in industrialized countries, might not be the optimal control tools in Africa. Control strategies should fit the needs and perceptions of local communities. Improved intersectoral and international collaboration in surveillance, diagnosis and control, and in the education of medical and veterinary personnel, are advocated.

Molasses as a possible cause of an "endocrine disruptive syndrome" in calves.

During the mid 1990s a potentially serious, chronic syndrome was reported in well-managed beef and dairy herds from unrelated parts of South Africa. Farmers reported that it manifested as various combinations of decreased production, decreased weaning masses, apparent immune breakdown in previously immunocompetent animals, increased reproductive disorders, various mineral imbalances in non-deficient areas and goitre, noticeable as enlarged thyroid glands. The farmers associated this syndrome with certain batches of sugar cane molasses and molasses-based products. The syndrome was reminiscent of an "endocrine disruptive syndrome". The objective of this study was to evaluate the suspected endocrine disruptive effect of molasses included in cattle feed. Using existing in vitro assays, four batches of molasses syrup were screened for possible inclusion in a calf feeding trial. Two batches were selected for the trial. Thirty-two, 4- to 6-week-old, weaned Holstein bull calves were included in the single phase, three treatment, parallel design experiment. In two of the groups of calves, two different batches of molasses were included in their rations respectively. The control group was fed a ration to which no molasses was added, but which was balanced for energy and mineral content. The mass gain of the calves was recorded over the 6-month study period. The calves were clinically examined every week and clinical pathology parameters, immune responses and endocrine effects were regularly evaluated. Even though endocrine disrupting effects were detected with the in vitro screening assays, these could not be reproduced in the calves in the experiment. The two batches of molasses utilized in the calf feeding trial did not induce major differences in any of the parameters measured, with the exception of a lower mass gain in one of the molasses-fed groups (Group 1), which tended towards significance. The results of the study indicate that the two batches of molasses had no endocrine disruptive or immunosuppressive effects in calves.

Evaluation of bone impingement prediction in pre-operative planning for shoulder arthroplasty.

In shoulder arthroplasty, malpositioning of prostheses often leads to reduced postoperative range of motion (ROM) and complications such as impingement, loosening, and dislocation. Furthermore, the risk of impingement complications increases when reverse total prostheses are used. For this purpose a pre-operative planning system was developed that enables surgeons to perform a virtual shoulder replacement procedure. The present authors' pre-operative planning system simulates patient-specific bone-determined ROM meant to reduce the risk of impingement complications and to improve the ROM of patients undergoing shoulder replacement surgery. This paper describes a validation experiment with the purpose of ratifying the clinical applicability and usefulness of the ROM simulation module for shoulder replacement surgery. The experiment was performed on cadaveric shoulders. A data connection was set up between the software environment and an existing intra-operative guidance system to track the relative positions of the bones. This allowed the patient-specific surface models to be visualized within the software for the position and alignment of the tracked bones. For both shoulders, ROM measurements were recorded and tagged with relevant information such as the type of prosthesis and the type of movement that was performed. The observed ROM and occurrences of impingement were compared with the simulated equivalents. The median deviation between observed impingement angles and simulated impingement angles was -0.30 degrees with an interquartile range of 5.20 degrees (from -3.40 degrees to 1.80 degrees). It was concluded that the ROM simulator is sufficiently accurate to fulfil its role as a supportive instrument for orthopaedic surgeons during shoulder replacement surgery.

Potential plant poisonings in dogs and cats in southern Africa.

Plant poisoning occurs less commonly in dogs and cats than in herbivorous livestock, but numerous cases have been documented worldwide, most of them caused by common and internationally widely cultivated ornamental garden and house plants. Few cases of poisoning of cats and dogs have been reported in southern Africa, but many of the plants that have caused poisoning in these species elsewhere are widely available in the subregion and are briefly reviewed in terms of toxic principles, toxicity, species affected, clinical signs, and prognosis. The list includes Melia azedarach (syringa), Brunfelsia spp. (yesterday, today and tomorrow), Datura stramonium (jimsonweed, stinkblaar), a wide variety of lilies and lily-like plants, cycads, plants that contain soluble oxalates, plants containing cardiac glycosides and other cardiotoxins and euphorbias (Euphorbia pulcherrima, E. tirucalli). Poisoning by plant products such as macadamia nuts, onions and garlic, grapes and raisins, cannabis (marijuana, dagga) or hashish and castor oil seed or seedcake is also discussed. Many of the poisonings are not usually fatal, but others frequently result in death unless rapid action is taken by the owner and the veterinarian, underlining the importance of awareness of the poisonous potential of a number of familiar plants.

Putative Aspergillus niger-induced oxalate nephrosis in sheep.

A sheep farmer provided a maize-based brewer's grain (mieliemaroek) and bales of Eragrostis curvula hay to ewes and their lambs, kept on zero-grazing in pens. The 'mieliemaroek' was visibly mouldy. After 14 days in the feedlot, clinical signs, including generalised weakness, ataxia of the hind limbs, tremors and recumbency, were noticed. Six ewes died within a period of 7 days. A post mortem examination was performed on 1 ewe. The carcass appeared to be cachectic with mild effusions into the body cavities; mild lung congestion and pallor of the kidneys were observed. Microscopical evaluation revealed nephrosis and birefringent oxalate crystals in the renal tubules when viewed under polarised light. A provisional diagnosis of oxalate nephrosis with subsequent kidney failure was made. Amongst other fungi, Aspergillus niger was isolated from 'mieliemaroek' samples submitted for fungal culture and identification. As A. niger is known to synthesise oxalates, a qualitative screen to detect oxalic acid in the mieliemaroek and purified A. niger isolates was performed using high-performance liquid chromatography (HPLC). Oxalic acid was detected, which supported a diagnosis of soluble oxalate-induced nephropathy.

Evaluation of activated charcoal as treatment for Yellow tulp (Moraea pallida) poisoning in cattle.

The efficacy of activated charcoal as a treatment for cattle (n = 57) poisoned by Yellow tulp (Moraea pallida) was investigated. Treatment with activated charcoal resulted in full recovery, irrespective of the degree of posterior paresis, provided that this clinical sign did not develop within the first 12 hours after initial exposure to Yellow tulp-infested grazing. For instance, despite treatment, 1 of 7 cattle succumbed after manifesting mild posterior paresis 6 to 8 h after initial exposure and 3 of 3 treated cattle died after developing severe posterior paresis within 6 to 12 h.

Putative neuromycotoxicoses in an adult male following ingestion of moldy walnuts.

A tremorgenic syndrome occurs in dogs following ingestion of moldy walnuts, and Penicillium crustosum has been implicated as the offending fungus. This is the first report of suspected moldy walnut toxicosis in man. An adult male ingested approximately eight fungal-infected walnut kernels and after 12 h experienced tremors, generalized pain, incoordination, confusion, anxiety, and diaphoresis. Following symptomatic and supportive treatment at a local hospital, the man made an uneventful recovery. A batch of walnuts (approximately 20) was submitted for mycological culturing and identification as well as for mycotoxin analysis. Penicillium crustosum Thom was the most abundant fungus present on walnut samples, often occurring as monocultures on isolation plates. Identifications were confirmed with DNA sequences. The kernels and shells of the moldy walnuts as well as P. crustosum isolates plated on yeast extract sucrose (YES) and Czapek yeast autolysate (CYA) agars and incubated in the dark at 25 °C for 7 days were screened for tremorgenic mycotoxins and known P. crustosum metabolites using a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method. A relatively low penitrem A concentration of only 1.9 ng/g was detected on the walnut kernels when compared to roquefortine C concentrations of 21.7 µg/g. A similar result was obtained from P. crustosum isolates cultured on YES and CYA, with penitrem A concentrations much lower (0.6-6.4 µg per g mycelium/agar) compared to roquefortine C concentrations (172-1225 µg/g). The authors surmised that besides penitrem A, roquefortine C might also play an additive or synergistic role in intoxication of man.

A xylem sap retrieval pathway in rice leaf blades: evidence of a role for endocytosis?

The structure and transport properties of pit membranes at the interface between the metaxylem and xylem parenchyma cells and the possible role of these pit membranes in solute transfer to the phloem were investigated. Electron microscopy revealed a fibrillar, almost tubular matrix within the pit membrane structure between the xylem vessels and xylem parenchyma of leaf blade bundles in rice (Oryza sativa). These pits are involved primarily with regulating water flux to the surrounding xylem parenchyma cells. Vascular parenchyma cells contain large mitochondrial populations, numerous dictyosomes, endomembrane complexes, and vesicles in close proximity to the pit membrane. Taken collectively, this suggests that endocytosis may occur at this interface. A weak solution of 5,6-carboxyfluorescein diacetate (5,6-CFDA) was applied to cut ends of leaves and, after a minimum of 30 min, the distribution of the fluorescent cleavage product, 5,6-carboxyfluorescein (5,6-CF), was observed using confocal microscopy. Cleavage of 5,6-CFDA occurred within the xylem parenchyma cells, and the non-polar 5,6-CF was then symplasmically transported to other parenchyma elements and ultimately, via numerous pore plasmodesmata, to adjacent thick-walled sieve tubes. Application of Lucifer Yellow, and, separately, Texas Red-labelled dextran (10 kDa) to the transpiration stream, confirmed that these membrane-impermeant probes could only have been offloaded from the xylem via the xylem vessel-xylem parenchyma pit membranes, suggesting endocytotic transmembrane transfer of these membrane-impermeant fluorophores. Accumulation within the thick-walled sieve tubes, but not in thin-walled sieve tubes, confirms the presence of a symplasmic phloem loading pathway, via pore plasmodesmata between xylem parenchyma and thick-walled sieve tubes, but not thin-walled sieve tubes.

Poisonous plants of veterinary and human importance in southern Africa.

Southern Africa is inherently rich in flora, where the habitat and climatic conditions range from arid environments to lush, sub-tropical greenery. Needless to say, with such diversity in plant life there are numerous indigenous poisonous plants, and when naturalised exotic species and toxic garden varieties are added the list of potential poisonous plants increases. The economically important poisonous plants affecting livestock and other plant poisonings of veterinary significance are briefly reviewed. In addition, a synopsis of the more common plant poisonings in humans is presented. Many of the plants mentioned in this review are also used ethnobotanically for treatment of disease in humans and animals and it is essential to be mindful of their toxic potential.

Provisional clinical chemistry parameters in the African sharptooth catfish (Clarias gariepinus).

Pollution affects aquatic systems worldwide and there is an urgent need for efficient monitoring. Fish are generally sensitive to their environment and are thus considered to be valuable bioindicator species. The African Sharptooth catfish (Clarias gariepinus) is particularly important in this respect because of its very wide distribution. In order to use C. gariepinus as a bioindicator species its baseline clinical chemistry must be defined. Existing data are scarce, and the objective of this work was therefore to establish clinical chemistry parameters for C. gariepinus. Blood was collected from male and female catfish and a number of clinical chemistry parameters were determined. Plasma protein values, but particularly those of plasma albumin, were found to be very low, approximately half the value for dogs, but similar to the values in Channel catfish (Ictalurus punctatus). Plasma urea values in Sharptooth catfish were found to be much lower than in dogs, but only marginally lower than in Channel catfish. Plasma creatinine in Sharptooth catfish, however, was only a quarter of that of dogs and one third of that found in Channel catfish. These findings may have implications for using urea and/or creatinine as an index of renal glomerular filtration, as is done in mammals. Plasma enzyme activity ranges were much lower in Sharptooth catfish than in dogs, particularly for alkaline phosphatase (ALP) and alanine aminotransferase (ALT). By comparison, Channel catfish have an even lower ALT activity range but an ALP range that is very similar to dogs. The implications for using these enzymes as markers for liver disease are not clear from these data, as factors such as plasma half-life and tissue distribution remain to be determined. The very low plasma thyroxine (T4) levels have important implications for laboratory personnel, who will have to set up calibration and standardisation adaptations for the methods that are generally designed for human samples. Although the sample size was too small for reliable comparisons, it appeared that there was little difference in the parameters measured between male and female fish. The values obtained are a useful starting point for using C. gariepinus as a bioindicator species.

Multimycotoxin analysis of South African Aspergillus clavatus isolates.

Aspergillus clavatus poisoning is a neuromycotoxicosis of ruminants that occurs sporadically across the world after ingestion of infected feedstuffs. Although various toxic metabolites are synthesized by the fungus, it is not clear which specific or group of mycotoxins induces the syndrome. A. clavatus isolates were deposited in the culture collection of the Biosystematics Division, Plant Protection Research Institute, Agricultural Research Council during incidences of livestock poisoning (1988-2016). Six isolates were still viable and these plus three other South African isolates that were also previously deposited in the collection were positively identified as A. clavatus based on morphology and ß-tubulin sequence data. The cultures were screened for multiple mycotoxins using a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method. Twelve A. clavatus metabolites were detected. The concentrations of the tremorgenic mycotoxins (i.e., tryptoquivaline A and its related metabolites deoxytryptoquivaline A and deoxynortryptoquivaline) were higher than patulin and cytochalasin E. Livestock owners should not feed A. clavatus-infected material to ruminants as all the South African A. clavatus isolates synthesized the same compounds when cultured under similar conditions.

Establishment and validation of primary hepatocytes of the African sharptooth catfish (Clarias gariepinus).

In vitro systems such as primary cells and continuous cell lines are gaining momentum in ecotoxicological studies. Cytotoxicity tests with fish cells as well as tests using specific endpoints such as CYP1A induction are valuable in the toxicity assessment of environmental samples. The main objective of this study was to establish and validate the use of primary hepatocytes from the African sharptooth catfish (Clarias gariepinus) as an in vitro toxicity monitoring system. The successful isolation of primary hepatocytes from the sharptooth catfish was achieved using an in situ perfusion method. The primary hepatocytes responded to CYP1A induction, while a continuous Chinese hamster ovary (CHO-K1) cell line showed no activity when exposed to various concentrations of benzo[a]pyrene (B[a]P) (p<0.0001). Cytotoxicity, as measured by the methyl thiazol tetrazolium (MTT) assay, was not observed following a 72 h exposure of the primary hepatocytes and the CHO-K1 cell line to different B[a]P concentrations. However, the hepatocytes were damaged at higher B[a]P concentrations (>10(-6)M) as shown by transmission electron microscopy. This cytotoxicity effect was also confirmed by the trypan blue exclusion assay (TD(50) of 10(-6)M). Differences in the results between the MTT and trypan blue exclusion assays are probably due to mitochondria that are still metabolically active, causing the tetrazolium salt to be dehydrogenated. The internal architecture of normal primary hepatocytes included large quantities of rough endoplasmic reticulum (often in close proximity to the nucleus), mitochondria, aggregates and scattered glycogen, a few lipid droplets and spherical nuclei with distinct nucleoli. The primary catfish hepatocyte cell culture system, expressing CYP1A when exposed to B[a]P, could be used as a biomarker for aromatic hydrocarbon pollutants in aquatic ecosystems of southern and East Africa.