Comprehensive analysis of a widely pharmaceutical, furosemide, and its degradation products in aquatic systems: Occurrence, fate, and ecotoxicity.

Many pharmaceutical compounds end up in the environment due to incomplete removal by wastewater treatment plants (WWTPs). Some compounds are sometimes present in significant concentrations and therefore represent a risk to the aquatic environment. Furosemide is one of the most widely used drugs in the world. Considered as an essential drug by the World Health Organization, this powerful loop diuretic is used extensively to treat hypertension, heart and kidney failure and many other purposes. However, this important consumption also results in a significant release of furosemide in wastewater and in the receiving environment where concentrations of a few hundred ng/L to several thousand have been found in the literature, making furosemide a compound of great concern. Also, during its transport in wastewater systems and WWTPs, furosemide can be degraded by various processes resulting in the production of more than 74 by-products. Furosemide may therefore present a significant risk to ecosystem health due not only to its direct cytotoxic, genotoxic and hepatotoxic effects in animals, but also indirectly through its transformation products, which are poorly characterized. Many articles classify furosemide as a priority pollutant according to its occurrence in the environment, its persistence, its elimination by WWTPs, its toxicity and ecotoxicity. Here, we present a state-of-the-art review of this emerging pollutant of interest, tracking it, from its consumption to its fate in the aquatic environment. Discussion points include the occurrence of furosemide in various matrices, the efficiency of many processes for the degradation of furosemide, the subsequent production of degradation products following these treatments, as well as their toxicity.

Deletion of Clusterin Protects Cochlear Hair Cells against Hair Cell Aging and Ototoxicity.

Hearing loss is a debilitating disease that affects 10% of adults worldwide. Most sensorineural hearing loss is caused by the loss of mechanosensitive hair cells in the cochlea, often due to aging, noise, and ototoxic drugs. The identification of genes that can be targeted to slow aging and reduce the vulnerability of hair cells to insults is critical for the prevention of sensorineural hearing loss. Our previous cell-specific transcriptome analysis of adult cochlear hair cells and supporting cells showed that Clu, encoding a secreted chaperone that is involved in several basic biological events, such as cell death, tumor progression, and neurodegenerative disorders, is expressed in hair cells and supporting cells. We generated Clu-null mice (C57BL/6) to investigate its role in the organ of Corti, the sensory epithelium responsible for hearing in the mammalian cochlea. We showed that the deletion of Clu did not affect the development of hair cells and supporting cells; hair cells and supporting cells appeared normal at 1 month of age. Auditory function tests showed that Clu-null mice had hearing thresholds comparable to those of wild-type littermates before 3 months of age. Interestingly, Clu-null mice displayed less hair cell and hearing loss compared to their wildtype littermates after 3 months. Furthermore, the deletion of Clu is protected against aminoglycoside-induced hair cell loss in both in vivo and in vitro models. Our findings suggested that the inhibition of Clu expression could represent a potential therapeutic strategy for the alleviation of age-related and ototoxic drug-induced hearing loss.

Sensorineural hearing loss may lead to dementia-related pathological changes in hippocampal neurons.

Presbycusis contributes to cognitive decline and Alzheimer's disease. However, most research in this area involves clinical observations and statistical modeling, and few studies have examined the relationship between hearing loss and the molecular changes that lead to cognitive dysfunction. The present study investigated whether hearing loss contributes to dementia in the absence of aging and noise using a mouse model of severe bilateral hearing loss induced by kanamycin (1000 mg/kg) and furosemide (400 mg/kg). Immunohistochemistry, silver staining, immunofluorescence analysis, and Western blotting were used to observe pathological changes in different regions of the hippocampus in animals with hearing loss. Changes in the cognitive function of animals with hearing loss were assessed using the Morris water maze test. The results showed that neurons began to degenerate 60 days after hearing loss, and this degeneration was accompanied by structural disorganization and decreased neurogenesis. The level of phosphorylated tau increased over time. Increases in escape latency and distance traveled during the training phase of the Morris water maze test were observed 90 days after hearing loss. Activated microglia and astrocytes with increased levels of inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) were detected in the hippocampus. These results suggest that hearing loss alone causes neuronal degeneration, inhibition of neurogenesis, increased tau protein phosphorylation, and increased neuroinflammation in the hippocampus. Early intervention in individuals with hearing loss may reduce the risk of cognitive decline.

Effects of combined gentamicin and furosemide treatment on cochlear ribbon synapses.

It is well-established that aminoglycoside antibiotics are ototoxic, and the toxicity can be drastically enhanced by the addition of loop diuretics, resulting in rapid irreversible hair cell damage. Using both electrophysiologic and morphological approaches, we investigated whether this combined treatment affected the cochlea at the region of ribbon synapses, consequently resulting in auditory synaptopathy. A series of varied gentamicin and furosemide doses were applied to C57BL/6 mice, and auditory brainstem responses (ABR) and distortion product otoacoustic emissions (DPOAE) were measured to assess ototoxic damage within the cochlea. In brief, the treatment effectively induced cochlear damage and promoted a certain reorganization of synaptic ribbons, while a reduction of ribbon density only occurred after a substantial loss of outer hair cells. In addition, both the ABR wave I amplitude and the ribbon density were elevated in low-dose treatment conditions, but a correlation between the two events was not significant for individual cochleae. In sum, combined gentamicin and furosemide treatment, at titrated doses below those that produce hair cell damage, typically triggers synaptic plasticity rather than a permanent synaptic loss.

miR-182 prevented ototoxic deafness induced by co-administration of kanamycin and furosemide in rats.

Ototoxic drugs may induce auditory sensory hair cell loss and permanent deafness; however, there is still no effective treatments or prevention strategies for this side effect. A recent study found that microRNA182 (miR-182) protected cochlear hair cells from ototoxic drug-induced apoptosis in vitro. However, it remains unclear whether miR-182 can protect drug-induced deafness in vivo. In this study, we overexpressed cochlear miR-182 in Sprague-Dawley rats by trans-round window niche delivery of miR-182 mimics. The rats subsequently received intraperitoneal injections of kanamycin and furosemide to induce acute cochlear outer hair cell death and permanent deafness. Auditory brainstem response tests showed that miR-182 attenuated permanent threshold shifts. Consistent with this result, miR-182 reduced the loss of outer hair cells and missing stereocilia. miR-182 treatment also increased the level of phosphoinositide-3 kinase regulatory subunit p85α in the outer hair cells after co-administration of kanamycin and furosemide. Our findings suggest that miR-182 has powerful protective potential against ototoxic drug-induced acute auditory sensory hair cell loss and permanent deafness.

WNK4 limits distal calcium losses following acute furosemide treatment.

The distal nephron is essential for calcium homeostasis. This is evidenced by disordered calcium transport following disrupted distal nephron function occurring in salt-wasting tubulopathies or with diuretic use. A plethora of studies support a role for WNK4 in thick ascending limb (TAL) and distal convoluted tubule ion transport with most studies focusing on sodium transport. Little is known about the in vivo role of WNK4 in regulating calcium homeostsis. Here, we investigated the role of WNK4 in regulating distal nephron calcium transport using WNK4 knockout animals (WNK4-/- ). As has been shown previously, we found that baseline urinary calcium levels are normal following WNK4 deletion. Following acute treatment with the loop diuretic, furosemide, which causes hypercalciuria through TAL inhibition, WNK4-/- animals demonstrated increased calcium wasting compared with wild-type controls. WNK4-/- animals had decreased TRPV5 expression along DCT2 supporting a mechanistic role for this calcium channel in the increased calciuresis. As this supported the hypothesis that WNK4-/- animals have a tendency toward calcium wasting under stress, we tested the effects of a calcium-deplete diet on urinary calcium excretion. Urinary calcium excretion and plasma ionized calcium levels were not different between control and knockout animals following consumption of a calcium-deplete diet. Our data show that WNK4, via regulation of TRPV5, limits distal calcium losses following acute treatment with furosemide; however, WNK4 deletion does not affect the chronic renal response to dietary calcium depletion. Our data reveal an in vivo role for WNK4 in distal nephron calcium handling that is important for fine-tuning calcium reabsorption.

Hearing consequences in Gjb2 knock-in mice: implications for human p.V37I mutation.

Human p.V37I mutation of GJB2 gene was strongly correlated with late-onset progressive hearing loss, especially among East Asia populations. We generated a knock-in mouse model based on human p.V37I variant (c.109G>A) that recapitulated the human phenotype. Cochlear pathology revealed no significant hair cell loss, stria vascularis atrophy or spiral ganglion neuron loss, but a significant change in the length of gap junction plaques, which may have contributed to the observed mild endocochlear potential (EP) drop in homozygous mice lasting lifetime. The cochlear amplification in homozygous mice was compromised, but outer hair cells' function remained unchanged, indicating that the reduced amplification was EP- rather than prestin-generated. In addition to ABR threshold elevation, ABR wave I latencies were also prolonged in aged homozygous animals. We found in homozygous IHCs a significant increase in ICa but no change in Ca2+ efficiency in triggering exocytosis. Environmental insults such as noise exposure, middle ear injection of KCl solution and systemic application of furosemide all exacerbated the pathological phenotype in homozygous mice. We conclude that this Gjb2 mutation-induced hearing loss results from 1) reduced cochlear amplifier caused by lowered EP, 2) IHCs excitotoxicity associated with potassium accumulation around hair cells, and 3) progression induced by environmental insults.

Acute body sodium depletion induces skin sodium mobilization in female Wistar rats.

NEW FINDINGS: What is the central question of this study? Can Na+ depletion mobilize Na+ from the skin reservoir in ovariectomized rats? Does oestrogen replacement change the amount and the dynamics of skin Na+ storage? Is the reduced salt appetite after Na+ depletion in ovariectomized rats with oestrogen replacement related to changes in the skin Na+ ? What is the main finding and its importance? This work demonstrated that acute body Na+ depletion induced by frusemide mobilized the osmotically inactive skin Na+ reservoir to become osmotically active. Oestrogen treatment decreased the induced Na+ intake in ovariectomized rats but did not modulate the inactive Na+ reservoir in control conditions or its mobilization induced by Na+ depletion. ABSTRACT: Oestradiol, which is an important hormone for water and electrolyte balance, also has a role in the inhibition of induced Na+ appetite. Sodium can be stored in the skin in osmotically active or inactive forms, and this skin Na+ reservoir may be involved in the control of body Na+ levels during physiopathological challenges. In this study, we investigated whether the effect of sodium depletion by frusemide can mobilize Na+ from the skin reservoir and whether oestradiol replacement changes or mobilizes the Na+ reserves in the skin. Ovariectomized Wistar rats were treated with vehicle or oestradiol for 7 days to evaluate the effects of oestrogen on the hydroelectrolyte balance, intake responses and skin Na+ and water content in basal conditions. Furthermore, the effects of oestrogen were evaluated after 24 h frusemide-induced whole-body Na+ depletion. Oestradiol-replaced rats exhibited reduced water intake without any significant changes in salt intake, Na+ excretion or water and Na+ skin content in basal conditions. After sodium depletion, both vehicle- and oestradiol-treated rats exhibited an increase in the osmotically active skin Na+ , which was associated with a decrease of the inactive skin Na+ reservoir. Oestrogen decreased the hypertonic saline intake induced by Na+ depletion, but it was not associated with any significant changes in the skin Na+ reservoir. Thus, sodium depletion is able to change the inactive-active skin Na+ reservoir balance. However, the oestrogenic modulation of sodium appetite after Na+ depletion is probably not related to the action of this hormone in the skin Na+ reservoir balance.

Generation of new hair cells by DNA methyltransferase (Dnmt) inhibitor 5-azacytidine in a chemically-deafened mouse model.

Regeneration of mature mammalian inner ear hair cells remains to be a challenge. This study aims to evaluate the ability of DNA methyltransferase (Dnmt) inhibitor 5-azacytidine (5-aza) to generate outer hair cells (OHCs) in a chemically-deafened adult mouse model. 5-aza was administrated into the mouse inner ear via the round window. Immunofluorescence was used to examine the expression of hair cell specific proteins following 5-aza treatment. The results showed that in the chemically-deafened mouse cochlea, new OHCs were found post 5-aza treatment, whereas OHCs were completely lost in saline-treated mice. New hair cells expressed multiple hair cell markers included Myosin VIIa, Pou4f3 and Myosin VI. Newly-generated hair cells presented in three cochlear turns and were able to survive for at least six weeks. The effects of new hair cells generation by 5-aza were concentration dependent. Quantitative PCR study indicates that 5-aza may function through Dnmt1 inhibition. The results of this report suggest that the Dnmt inhibitor 5-aza may promote hair cell regeneration in a chemically-deafened mouse model.

Vitamin C alleviates ototoxic effect caused by coadministration of amikacin and furosemide.

BACKGROUND: Drug-induced ototoxicity is still a main clinical problem in otolaryngology. It is widely known that aminoglycoside antibiotics combined with loop diuretics significantly contribute to permanent ototoxicity. The aim of this study was to find out whether ascorbic acid (vitamin C) is able to reverse or alleviate ototoxicity evoked by systemic (ip) administration of combination of amikacin and furosemide in experimental male albino Swiss mice. METHODS: Ototoxic combination of amikacin and furosemide was isobolographically evaluated based on the hearing threshold decreasing doses by 20% and 50% (TDD20 and TDD50), respectively. Linear regression analysis was used to determine the TDD20 and TDD50 values for amikacin, furosemide, vitamin C administered alone and in combination (at the fixed-ratio of 1:1). RESULTS: Vitamin C (in a dose of 500 mg/kg, ip) alleviated the impairment in hearing threshold evoked by combined ip administration of amikacin and furosemide (at the fixed-ratio of 1:1) in mice by reducing TDD50 values from 49.82 to 21.56 (p < 0.01). In contrast, vitamin C (500 mg/kg, ip) had no significant effect on TDD20 values for the combination of amikacin and furosemide at the fixed-ratio of 1:1. CONCLUSIONS: Vitamin C administered together with ototoxic drug combination of amikacin and furosemide reduced ototoxicity evoked by this two-drug combination in the experimental mice.

Potassium-sparing effects of furosemide in mice on high-potassium diets.

In individuals on a regular "Western" diet, furosemide induces a kaliuresis and reduction in plasma K concentration by inhibiting Na reabsorption in the thick ascending limb of Henle's loop, enhancing delivery of Na to the aldosterone-sensitive distal nephron. In the aldosterone-sensitive distal nephron, the increased Na delivery stimulates K wasting due to an exaggerated exchange of epithelial Na channel-mediated Na reabsorption of secreted K. The effects of furosemide are different in mice fed a high-K, alkaline (HK) diet: the large-conductance Ca-activated K (BK) channel, in conjunction with the BK ß4-subunit (BK-α/ß4), mediates K secretion from intercalated cells (IC) of the connecting tubule and collecting ducts. The urinary alkaline load is necessary for BK-α/ß4-mediated K secretion in HK diet-fed mice. However, furosemide acidifies the urine by increasing vacuolar ATPase expression and acid secretion from IC, thereby inhibiting BK-α/ß4-mediated K secretion and sparing K. In mice fed a low-Na, high-K (LNaHK) diet, furosemide causes a greater increase in plasma K concentration and reduction in K excretion than in HK diet-fed mice. Micropuncture of the early distal tubule of mice fed a LNaHK diet, but not a regular or a HK diet, reveals K secretion in the thick ascending limb of Henle's loop. The sites of action of K secretion in individuals consuming a high-K diet should be taken into account when diuretic agents known to waste K with low or moderate K intakes are prescribed.

Mkp-1 protects mice against toxin-induced liver damage by promoting the Nrf2 cytoprotective response.

The present study was undertaken to investigate the possible protective effect of mitogen-activated protein kinase phosphatase 1 (Mkp-1) on toxin-induced hepatic injury. Here, we uncovered a positive feedback loop between Mkp-1, a dual threonine/tyrosine phosphatase, and nuclear factor erythroid 2-related factor 2 (Nrf2), a crucial regulator of the defense system in the liver. Mkp-1-/- mice exhibited decreased protein levels of Nrf2, phase II gene products, and reduced glutathione (GSH) in the liver. Induction of detoxifying enzymes by the Nrf2 activator butylated hydroxyanisole (BHA) or sulforaphane, was attenuated in the liver and small intestines of Mkp-1-/- mice, indicating that the Nrf2 signaling pathway is impaired as a result of Mkp-1 deficiency. Mkp-1-/- mice suffered more severe liver injury after a single exposure to hepatotoxin carbon tetrachloride (CCl4) than their wild-type (WT) counterparts. BHA partially rescued the CCl4-induced liver damage in WT mice, but not in Mkp-1-/- mice, suggesting the requirement of Mkp-1 in the activation of Nrf2 signaling against the liver injury. Mechanistically, Mkp-1 upregulated Nrf2 through a direct interaction with the Neh2 domain in the transcription factor, while Nrf2 enhanced the expression of Mkp-1 mRNA by binding to the ARE site at -1719 to -1710bp in the Mkp-1 promoter. Our results reveal novel role of Mkp-1 in the maintenance of redox homeostasis in the liver. Thus, strategies aimed at augmenting Mkp-1 expression may be beneficial in protecting the liver and may provide novel therapeutic approaches to toxin-induced liver injury.

Genetic disruption of fractalkine signaling leads to enhanced loss of cochlear afferents following ototoxic or acoustic injury.

Cochlear hair cells are vulnerable to a variety of insults like acoustic trauma and ototoxic drugs. Such injury can also lead to degeneration of spiral ganglion neurons (SGNs), but this occurs over a period of months to years. Neuronal survival is necessary for the proper function of cochlear prosthetics, therefore, it is of great interest to understand the mechanisms that regulate neuronal survival in deaf ears. We have recently demonstrated that selective hair cell ablation is sufficient to attract leukocytes into the spiral ganglion, and that fractalkine signaling plays a role in macrophage recruitment and in the survival of auditory neurons. Fractalkine (CX3 CL1), a chemokine that regulates adhesion and migration of leukocytes is expressed by SGNs and signals to leukocytes via its receptor CX3 CR1. The present study has extended the previous findings to more clinically relevant conditions of sensorineural hearing loss by examining the role of fractalkine signaling after aminoglycoside ototoxicity or acoustic trauma. Both aminoglycoside treatment and acoustic overstimulation led to the loss of hair cells as well as prolonged increase in the numbers of cochlear leukocytes. Lack of CX3 CR1 did not affect macrophage recruitment after injury, but resulted in increased loss of SGNs and enhanced expression of the inflammatory cytokine interleukin-1ß, when compared to mice with intact CX3 CR1. These data indicate that the dysregulation of macrophage response caused by the absence of CX3 CR1 may contribute to inflammation-mediated neuronal loss in the deafened ear, suggesting a key role for inflammation in the long-term survival of target-deprived afferent neurons.

Scar formation in mice deafened with kanamycin and furosemide.

In mammals, hair cell loss is irreversible and leads to hearing loss. To develop and test the functioning of different strategies aiming at hair cell regeneration, animal models of sensorineural hearing loss are essential. Although cochleae of these animals should lack hair cells, supporting cells should be preserved forming an environment for the regenerated hair cells. In this study, we investigated how ototoxic treatment with kanamycin and furosemide changes the structure of cochlear sensory epithelium in mice. The study also compared different tissue preparation protocols for scanning electron microscopy (SEM). Cochleae were collected from deafened and nondeafened mice and further processed for plastic mid modiolar sections and SEM. For comparing SEM protocols, cochleae from nondeafened mice were processed using three protocols: osmium-thiocarbohydrazide-osmium (OTO), tannic acid-arginine-osmium, and the conventional method with gold-coating. The OTO method demonstrated optimal cochlear tissue preservation. Histological investigation of cochleae of deafened mice revealed that the supporting cells enlarged and ultimately replaced the lost hair cells forming types 1 and 2 phalangeal scars in a base towards apex gradient. The type 3 epithelial scar, flattened epithelium, has not been seen in analysed cochleae. The study concluded that mice deafened with kanamycin and furosemide formed scars containing supporting cells, which renders this mouse model suitable for testing various hair cell regeneration approaches. Microsc. Res. Tech. 79:766-772, 2016. © 2016 Wiley Periodicals, Inc.

A coupled Bio-EF process for mineralization of the pharmaceuticals furosemide and ranitidine: Feasibility assessment.

A coupled Bio-EF treatment has been applied as a reliable process for the degradation of the pharmaceuticals furosemide (FRSM) and ranitidine (RNTD) in aqueous medium, in order to reduce the high energy consumption related to electrochemical technology. In the first stage of this study, electrochemical degradation of the drugs was assessed by the electro-Fenton process (EF) using a BDD/carbon-felt cell. Biodegradability of the drugs solutions was enhanced reaching BOD5/COD ratios close to the biodegradability threshold of 0.4, evidencing the formation of bio-compatible by-products (mainly short-chain carboxylic acids) which are suitable for biological post-treatment. Moreover, toxicity evaluation by the Microtox(®) method revealed that EF pre-treatment was able of detoxifying both, FRSM and RNTD solutions, constituting another indicator of biodegradability of EF treated solutions. In the second stage, electrolyzed solutions were treated by means of an aerobic biological process. A significant part of the short-chain carboxylic acids formed during the electrochemical phase was satisfactorily removed by the used selected microorganisms. The results obtained demonstrate the efficiency and feasibility of the integrated Bio-EF process.

The role of intramolecular self-destruction of reactive metabolic intermediates in determining toxicity.

When reactive centers are formed in chemical conversions, intermolecular reactions tend to dominate over intramolecular alternatives whenever both alternatives are possible. Hence, when reactive metabolites are formed from xenobiotics, intramolecular quenching by moieties adjacent to a toxicophore may play an important role in reducing toxicity related to reactive intermediates. The phenomenon is likely to be particularly noticeable for toxicophores that are readily associated with a type of toxicity that is rarely caused by other structural motives. In two demonstrative investigations, it is concluded that nitrobenzenes for which the expected nitrosyl metabolite is likely to react with adjacent groups are less toxic than what is rationally expected, and that among aryl amine drugs allowing for the immediate quenching of the corresponding N-aryl hydroxylamine metabolite, the typical erythrocyte toxicity often seen with aryl amines is absent. The deliberate introduction of effective quenching groups nearby a toxicophoric moiety may present a potential strategy for reducing toxicity in the design of drugs and other man-made xenobiotics.

Effect of Cyclooxygenase(COX)-1 and COX-2 inhibition on furosemide-induced renal responses and isoform immunolocalization in the healthy cat kidney.

BACKGROUND: The role of cyclooxygenase(COX)-1 and COX-2 in the saluretic and renin-angiotensin responses to loop diuretics in the cat is unknown. We propose in vivo characterisation of isoform roles in a furosemide model by administering non-steroidal anti-inflammatory drugs (NSAIDs) with differing selectivity profiles: robenacoxib (COX-2 selective) and ketoprofen (COX-1 selective). RESULTS: In this four period crossover study, we compared the effect of four treatments: placebo, robenacoxib once or twice daily and ketoprofen once daily concomitantly with furosemide in seven healthy cats. For each period, urine and blood samples were collected at baseline and within 48 h of treatment starting. Plasma renin activity (PRA), plasma and urinary aldosterone concentrations, glomerular filtration rate (GFR) and 24 h urinary volumes, electrolytes and eicosanoids (PGE2, 6-keto-PGF1α, TxB2), renal injury biomarker excretions [N-acetyl-beta-D-glucosaminidase (NAG) and Gamma-Glutamyltransferase] were measured. Urine volume (24 h) and urinary sodium, chloride and calcium excretions increased from baseline with all treatments. Plasma creatinine increased with all treatments except placebo, whereas GFR was significantly decreased from baseline only with ketoprofen. PRA increased significantly with placebo and once daily robenacoxib and the increase was significantly higher with placebo compared to ketoprofen (10.5 ± 4.4 vs 4.9 ± 5.0 ng ml(-1) h(-1)). Urinary aldosterone excretion increased with all treatments but this increase was inhibited by 75 % with ketoprofen and 65 % with once daily robenacoxib compared to placebo. Urinary PGE2 excretion decreased with all treatments and excretion was significantly lower with ketoprofen compared to placebo. Urinary TxB2 excretion was significantly increased from baseline only with placebo. NAG increased from baseline with all treatments. Immunohistochemistry on post-mortem renal specimens, obtained from a different group of cats that died naturally of non-renal causes, suggested constitutive COX-1 and COX-2 co-localization in many renal structures including the macula densa (MD). CONCLUSIONS: These data suggest that both COX-1 and COX-2 could generate the signal from the MD to the renin secreting cells in cats exposed to furosemide. Co-localization of COX isoenzymes in MD cells supports the functional data reported here.

High-content screening imaging and real-time cellular impedance monitoring for the assessment of chemical's bio-activation with regards hepatotoxicity.

Testing hepatotoxicity is a crucial step in the development and toxicological assessment of drugs and chemicals. Bio-activation can lead to the formation of metabolites which may present toxicity for the organism. Classical cytotoxic tests are not always appropriate and are often insufficient, particularly when non metabolically-competent cells are used as the model system, leading to false-positive or false-negative results. We tested over 24 h the effects of eight reference compounds on two different cell models: primary cultures of rat hepatocytes and FAO hepatoma cells that lack metabolic properties. We performed inter-assay validation between three classical cytotoxicity assays and real-time cell impedance data. We then complemented these experiments with high-content screening (HCS) to determine the cell function disorders responsible for the observed effects. Among the different assays used, the neutral red test seemed to be well suited to our two cell models, coupled with real-time cellular impedance which proved useful in the detection of bio-activation. Indeed, impedance monitoring showed a high sensitivity with interesting curve profiles yet seemed unsuitable for evaluation of viability on primary culture. Finally, HCS in the evaluation of hepatotoxicity is likely to become an essential tool for use in parallel to a classical cytotoxic assay in the assessment of drugs and environmental chemicals.

Humanized thymidine kinase-NOG mice can be used to identify drugs that cause animal-specific hepatotoxicity: a case study with furosemide.

Interspecies differences have limited the predictive utility of toxicology studies performed using animal species. A drug that could be a safe and effective treatment in humans could cause toxicity in animals, preventing it from being used in humans. We investigated whether the use of thymidine kinase (TK)-NOG mice with humanized livers could prevent this unfortunate outcome (i.e., "rescue" a drug for use in humans). A high dose of furosemide is known to cause severe liver toxicity in mice, but it is a safe and effective treatment in humans. We demonstrate that administration of a high dose of furosemide (200 mg/kg i.p.) causes extensive hepatotoxicity in control mice but not in humanized TK-NOG mice. This interspecies difference results from a higher rate of production of the toxicity-causing metabolite by mouse liver. Comparison of their survival curves indicated that the humanized mice were more resistant than control mice to the hepatotoxicity caused by high doses of furosemide. In this test case, humanized TK-NOG mouse studies indicate that humans could be safely treated with a high dose of furosemide.

Characterization of human lymphoblastoid cell lines as a novel in vitro test system to predict the immunotoxicity of xenobiotics.

Evaluating immunomodulatory effects of xenobiotics is an important component of the toxicity studies. Herein we report on the establishment of a novel invitro test system for the immunotoxicity screening of xenobiotics based on human lymphoblastoid cell lines (LCLs). Four immunotoxic compounds; tributyltin chloride, cyclosporine A, benzo(a)pyrene and verapamil hydrochloride, as well as three immune-inert compounds; urethane, furosemide and mannitol were selected for characterization. The treatment of LCLs with immunosuppressive compounds resulted in reduced viability. The IC50 values determined in human LCLs were in agreement with the data obtained for human peripheral mononuclear cells. Since cytokine production reflects lymphocytes responses to external stimuli, we evaluated the functional responses of LCLs by monitoring their pro-inflammatory and immunoregulatory cytokine production. Our findings prove that LCLs allowed for reliable differentiation between immunomodulatory and immune-inert compounds. Hence, pre-treatment with immunomodulatory compounds led to a decrease in the production of pro-inflammatory TNFα, IL-6 and immunoregulatory IL-2, IL-4, IL-10 and IFNγ cytokines, when compared to untreated ionomycin/PMA stimulated cells. Moreover, testing a panel of ten LCLs derived from unrelated healthy individuals reflects inter-individual variability in response to immunomodulatory xenobiotics. In conclusion, LCLs provide a novel alternative method for the testing of the immunotoxic effects of xenobiotics.