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PubMed1997

Action of peripheral or intrathyroidal lymphocytes on autologous thyrocytes cultured in follicles in collagen gel.

We have studied the action of peripheral blood lymphocytes (PBLs) and intrathyroidal lymphocytes (ITLs) on the biochemical and hormonal metabolism of autologous thyrocytes cultured in follicles in a collagen gel. The production of tumour necrosis factor alpha (TNF-alpha) in culture was also measured. Thyroid tissues and lymphocytes were obtained from ten patients with Graves' disease and from five control subjects. Lymphocyte-induced cytotoxicity was evaluated in autologous thyrocytes cultured in a collagen gel by several tests; neutral red uptake, lactate dehydrogenase activity and glutathione level. Hormonal metabolism was assessed by evaluating tri-iodothyronine (T3) and total cAMP production under TSH stimulation. TNF-alpha levels were measured in supernatants after 5 days of coculture. PBLs altered biochemical metabolism, T3 synthesis and cAMP production in autologous thyroid follicles. These inhibitions were greater than those obtained with ITLs. No difference was seen between cells obtained from patients with Graves' disease and those from normal subjects. TNF-alpha levels secreted by PBLs were higher than those secreted by ITLs. The concentrations of this cytokine decreased in coculture. Significant correlations were observed between the decrease in biochemical and hormonal parameters and TNF-alpha levels. Exogenous TNF-alpha and high doses of interferon gamma inhibited follicle metabolism, especially hormone secretion. In conclusion, thyrocytes cultured in follicles provide a more sensitive model than monolayer cultures for analysis of lymphocyte-induced interactions. Lymphocytes gradually inhibit the biochemical and hormonal metabolism of autologous thyroid follicles depending on the isolation method. These alterations may be particularly attributed to TNF-alpha secreted by lymphocytes. The cytokine-induced inhibition of thyroid hormonal function apparently involves the adenylate cyclase system.

Adult↗
PubMed1998

New method to measure the retention of viscoelastic agents on a rabbit corneal endothelial cell line after irrigation and aspiration.

PURPOSE: To quantify the surface retention of several ophthalmic viscoelastic agents following irrigation and aspiration (I/A) using a new in vitro method. SETTING: Alcon Laboratories, Inc., Fort Worth, Texas, USA. METHODS: A rabbit corneal endothelial cell line was cultured to confluency in 24-well plates, and the cells were labeled quantitatively with internalized neutral red dye. Five ophthalmic viscoelastic agents were applied to cover the monolayer of cells: sodium hyaluronate (Healon, Provisc, and Amvisc Plus), Formulation A (a dispersive, nonproteinaceous, synthetic polymer), and sodium chondroitin sulfate, sodium hyaluronate (Viscoat). Irrigation and aspiration (with fluid turbulence similar to that encountered in phacoemulsification surgery) were performed on each well for 3 minutes, using 120 mL of balanced salt solution with bicarbonate. dextrose, and glutathione (BSS Plus). The cells were treated with an acidified ethanol solution to extract the dye from the cells left without a viscoelastic cover. The extracted dye was measured by spectrophotometry and compared with the total dye recovered from control cells. RESULTS: The retention value, which represented the percentage of cells with viscoelastic retained on the surface, was calculated as follows: Healon, 7; Provisc, 16; Amvisc Plus, 17; Formulation A, 55; Viscoat, 90. On a nonadsorptive, non-cell surface, the retention values of the five viscoelastics were significantly less than those on cells. CONCLUSION: The results of this experimental model suggest that cohesive viscoelastics are readily removed from the cells, while dispersive viscoelastics are highly retained. In addition, mutual surface interaction (electrical charge and other properties) plays a significant role in determining the retention of viscoelastics on the corneal endothelial cell surface following I/A.

Animals↗
PubMed1996

Morphology of ricin and abrin exposed endothelial cells is consistent with apoptotic cell death.

Cultures of bovine pulmonary endothelial (BPE) cells were exposed to LC70 doses of ricin or abrin (15.5 and 4.5 pM respectively) over a period of up to 40 h. The viability of the cultures (as determined by the neutral red (NR) dye retention assay) declined after 6 h exposure to the toxins. From 15 h onwards, cellular material in toxin exposed cultures became detached from the substratum of the culture vessels. Hoffman modulation contrast photomicrography showed that this process was due to ricin and abrin exposed cells collapsing into membrane bound vesicles which retained the NR dye, became detached and floated into the medium. These apoptotic-like structural changes were further investigated by transmission electron microscopy (TEM) and by agarose gel electrophoresis of DNA from control and exposed cultures. Many of the characteristic changes associated with apoptotic cell death were seen using TEM, including heterochromatin condensation at the nuclear periphery, crenulation of the nuclear membrane and progressive degeneration of residual nuclear and cytoplasmic structures. The plasma membrane of many cells remained intact, and contained nuclear and cytoplasmic debris. Agarose gel electrophoresis of DNA extracted from toxin-treated cells revealed oligonucleosome sized DNA fragments, characteristic of apoptosis, from adherent cells at 7 h and both adherent and floating populations when harvested from 15 h; DNA from unexposed control cells did not show this fragmentation. The identification of apoptosis as being a significant additional mechanism of toxicity following exposure to ricin and abrin holotoxins raises the possibility of developing new therapeutic strategies against poisoning by these phytotoxins.

Abrin↗
PubMed1995

Regenerated nerve fibers in the noise-damaged chinchilla cochlea are not efferent.

Nerve-fiber regeneration in the chinchilla cochlea following a traumatic noise exposure was systematically described by Bohne and Harding (1992). However, their study did not determine the origin of the regenerated nerve fibers (RNFs). In the present study, 23 chinchillas were exposed for 12 h to a 0.5 kHz octave band of noise at 120 dB SPL. After a 3-month or 1-year recovery period, their right cochleas were incubated to demonstrate acetylcholinesterase (AChE) activity and then briefly counterstained with Neutral Red or OsO4. Their left cochleas were fixed with OsO4 and dissected using a combined organ of Corti (OC)/modiolus technique that preserved both structures for high-resolution microscopy. All cochleas were prepared as plastic-embedded flat preparations. Damage was located in the basal two-thirds of the cochlea and generally consisted of multiple lesions in the OC, often involving total degeneration of one or more OC segments (i.e., OC wipeouts). The OC wipeouts were separated from one another by areas which contained some identifiable cells of the OC (i.e., OC remnants). Most RNFs were found in OC wipeouts adjacent to OC remnants. In those animals (83%) with significant OC damage, 13 (100%) 3-month-recovery chinchillas had 1-96 RNFs while 6 (86%) 1-year-recovery chinchillas had 7-62 RNFs. In the AChE-stained cochleas, none of the RNFs were AChE-positive, but normal AChE-positive fibers were found in the undamaged apical turn. A variable number of surviving spiral ganglion cells was present in those regions of Rosenthal's canal that had originally innervated the missing hair cells in the OC wipeouts and remnants. It is concluded that RNFs are not part of the efferent cochlear system and therefore, most likely belong to the afferent system.

Acetylcholinesterase↗
PubMed2001

Quantitative assessment of primary skin irritants in vitro in a cytotoxicity model: comparison with in vivo human irritation tests.

BACKGROUND: While great efforts have been made in recent years to develop in vitro methods for assessing skin irritation potential, there are relatively few data that correlate in vitro data with in vivo data. OBJECTIVES: To expand our previously reported investigations on in vitro vs. in vivo correlation of a series of homologous N-alkyl sulphates of different alkyl chain length to include primary skin irritants of different chemical classes. METHODS: Anionic surfactants (three different sodium alkyl sulphonates and sodium lauryl sulphate), cationic surfactants (three alkyl trimethyl ammonium bromides), non-ionic surfactants (polyoxyethylene-20-cetyl ether and Tween 20), benzoic acid, dimethyl sulphoxide and phenol were chosen as model irritants. A spontaneously immortalized human keratinocyte line, HaCaT, was used as an in vitro model to predict the cutaneous irritation. The end-point used to assess toxicity was uptake of the vital dye neutral red (NR) 24 h after dosing. The cytotoxicity data from these assays were compared with the irritant responses (as evaluated by measurement of erythema and transepidermal water loss) obtained after 24-h application of the same compounds (100 microL of 20 mmol L(-1) aqueous solution) to the volar forearm of human volunteers. RESULTS: All tested irritants had cytotoxic effects as demonstrated by a decreased NR uptake, which showed a clear dose-response relationship. Concentrations resulting in 50% inhibition of NR uptake (IC(50)) ranged from 8 micromol L(-1) (hexadecyl trimethyl ammonium bromide) to 328 mmol L(-1) (dimethyl sulphoxide). We found a good overall correlation between in vitro cytotoxicity (NR uptake IC(50) values) and in vivo irritation potential in humans. Only the high molecular weight compounds Tween 20 and polyoxyethylene-20-cethyl ether were problematic, as their irritation potential was overestimated by the in vitro assay. This non-conformity of these high molecular weight (> 1000) compounds was expected, and can be largely attributed to the epidermal permeability barrier. The epidermal barrier, which greatly limits the percutaneous penetration of xenobiotics in vivo, does not exist in cell culture models. CONCLUSIONS: The in vitro cytotoxicity model is a useful screening tool, but data should be interpreted critically and require confirmation by appropriate in vivo studies.

Cell Culture Techniques↗
PubMed2000

Toxic evaluation of organic extracts from airborne particulate matter in Puerto Rico.

In recent years, several hypotheses have emerged to explain the toxicologic activity of particulate matter. Organic compounds, ultrafine particles, biologic components, and transition metals are some of the constituents that reportedly exert some type of adverse effect on human health. A considerable fraction of the urban particulate matter consists of carbon compounds, which originate mostly from anthropogenic sources. The toxicity of organic fractions from particulate matter have been mainly evaluated by considering their mutagenic activity. This research expands on the toxicologic profile of organic compounds adsorbed to particulate matter, specifically in Puerto Rico, by using the cytotoxic neutral red bioassay (NRB). The NRB uses normal human epidermal keratinocytes or other types of cells to measure the effect on cell viability when exposed to organic compounds associated to the particles in the air. We validated the NRB for particulate matter by using a standard reference material (SRM 1649). We used the NRB to determine toxicologic differences of extracts between an urban industrialized site with anthropogenic activity versus a coastal region with less human activity. The cytotoxicity associated with organic compounds in particulate matter collected at the urban industrialized site was detected in both the particulate matter (3/4) 10 microm in aerodynamic diameter (PM(10)) and particulate matter (3/4) 100 microm in aerodynamic diameter (PM(100)). Greater toxic effects were observed in PM(10) extracts than in PM(100) extracts, but PM(10) toxic effects were not significantly different from those in PM(100). The extracts from the industrialized site were more cytotoxic than the extracts from coastal reference site, although in the summer, extracts from both sites were significantly cytotoxic to normal human epidermal keratinocytes. In addition, the nonpolar extracts of both PM(10) and PM(100) exerted the greatest cytotoxicity, followed by the polar, and, finally, the moderately polar extract. This study demonstrates that extracts from the Guaynabo industrialized site were more toxic than similar extracts obtained from a reference coastal site in Fajardo, Puerto Rico.

Air Pollutants↗
PubMed2002

Changes in glutathione-redox balance induced by hexachlorocyclohexane and lindane in CHO-K1 cells.

1. The basal cytotoxic effect of the organochlorine pesticides hexachlorocyclohexane and lindane on CHO-K1 cultures was assessed at fractions of their lethal doses as determined by the neutral red incorporation (NRI) assay (NRI(6.25), NRI(12.5) and NRI(25)). The sulphur-redox cycle enzymes glutathione peroxidase, glutathione reductase and glutathione S-transferase, and total and oxidized glutathione were evaluated at several points during the standard growth curve of the cultures. 2. After incubation with each compound for 24 h, both glutathione peroxidase and reductase showed a substantial increase at the lowest exposure doses (NRI(6.25))--more significantly for lindane than for 1,2,3,4,5,6-hexachlorocyclohexane (HCH)--and dropped at higher doses of both compounds. The reduced and oxidized glutathione content was greatly diminished at the lower exposures, whereas the total glutathione content was higher at NRI(12.5) values. 3. Changes in cell membrane integrity were assessed for a wide range of pesticide concentrations with the lactate dehydrogenase release assay and lipid peroxidation. Membrane leakage and peroxide production were significantly enhanced at concentrations of HCH 50 microg ml(-1), although this effect was not significant at lindane concentrations < 200 microg ml(-1). 4. Lipid peroxidation increased with exposure to HCH at concentrations as low as NRI(6.25), whereas in the case of lindane, this increase was only significant at doses of NRI(25) and above.

Animals↗
PubMedDate not supplied

Evaluation of tissue and cellular biomarkers to assess 2,4,6-trinitrotoluene (TNT) exposure in earthworms: effects-based assessment in laboratory studies using Eisenia andrei.

The lysosomal neutral red retention time (NRRT) assay, a biomarker for lysosomal membrane stability, and the total immune activity (TIA) assay, a measure of non-specific immune system activity, were used in laboratory studies to assess the toxic effects of 2,4,6-trinitrotoluene (TNT) on earthworms (Eisenia andrei) in vivo. The results were compared with the concentration of TNT and its metabolites in earthworm tissue, as well as standard sublethal toxicity endpoints including growth (i.e. weight change) and reproduction effects from previously published studies. Filter paper experiments indicated a significant decrease in NRRT at >or=1.8 micro g TNT cm(-2), whereas sublethal (weight loss) and lethal effects to earthworms were detected at >or=3.5 and 7.1 micro g TNT cm(-2), respectively. Experiments in artificial soil showed that NRRT effects could be detected at lower TNT concentrations (>or=55 mg TNT kg(-1) soil dry weight) compared with other sublethal endpoints (effects on growth and reproduction). The TIA biomarker did not significantly respond to TNT. Copper (as CuSO4, filter paper contact tests) and 2-chloroacetamide (soil tests), which were used as reference toxicants, also decreased the NRRT. The use of the NRRT assay linked with tissue concentrations of TNT metabolites in earthworms was identified as a potentially appropriate biomarker approach for TNT exposure assessment under laboratory conditions and a novel tool for effects-based risk assessment.

Animals↗
PubMed1996

Benzoyl peroxide cytotoxicity evaluated in vitro with the human keratinocyte cell line, RHEK-1.

The human keratinocyte cell line, RHEK-1, was used to evaluate the cytotoxicity of benzoyl peroxide (BZP). As determined with the neutral red (NR) cytotoxicity assay, the 24-h midpoint (NR50) toxicity values, in mM, were 0.11 for BZP and 29.5 for benzoic acid, the stable metabolite of BZP. Irreversible cytotoxicity occurred after a 1-h exposure to 0.15 mM BZP and greater. When exposed to BZP for 7 days, a lag in growth kinetics was first observed at 0.06 mM BZP. Damage to the integrity of the plasma membrane was evident, as leakage of lactic acid dehydrogenase occurred during a 4-h exposure to BZP at 0.05 mM and greater. Intracellular membranes were also affected, as extensive vacuolization, initially perinuclear but then spreading throughout the cytoplasm, was noted in BZP-stressed cells. The generation of reactive free radicals from BZP was suggested by the following: the intracellular content of glutathione was lowered in cells exposed to BZP; cells pretreated with the glutathione-depleting agent, chlorodinitrobenzene, were hypersensitive to a subsequent challenge with BZP; lipid peroxidation by BZP was inducible in the presence of Fe2+; and cells previously maintained in a medium amended with vitamin E, an antioxidant, were more resistant to BZP, showed less lipid peroxidation in the presence of BZP+Fe2+ and did not develop the extensive intracellular vacuolization as compared to non-vitamin E maintained cells.

Benzene Derivatives↗
PubMed1985

Ionic permeabilities of the plasma membrane of isolated intact bovine rod outer segments as studied with a novel optical probe.

The permeability properties of the plasma membrane of intact rod outer segments purified from bovine retinas (ROS) were studied with the aid of the optical probe neutral red as described in the companion paper. The following observations were made: Electrical shunting of ROS membranes greatly stimulated Na+ and K+ transport, suggesting that this transport reflects Na+ and K+ currents, respectively. The dissipation of a Na+ gradient across the plasma membrane occurred with a half-time of 30 sec at 25 degrees C. The Na+ permeability was progressively inhibited when the external Ca2+ concentration was raised from 1 microM to 20 mM. A similar Ca2+ dependence was observed for H+ and Li+ transport. The Na+ permeability was not affected when the total internal Ca2+ content of ROS was varied between 0.1 mol Ca2+/mol rhodopsin and 7 mol Ca2+/mol rhodopsin, or when the free internal Ca2+ concentration was varied between 0.1 and 50 microM. The K+ permeability was progressively stimulated when the external Ca2+ concentration was raised from 0.001 to 1 microM, whereas a further increase to 20 mM was without effect. A similar Ca2+ dependence was observed for Rb+ and Cs+ transport. At an external Ca2+ concentration in the micromolar range the rate of transport decreased in the order: Na+ greater than K+ = H+ greater than Cs+ greater than Li+. Na+ fluxes depended in a sigmoidal way on the external Na+ concentration, suggesting that sodium ions move in pairs. The concentration dependence of uniport Na+ transport and that of Na+-stimulated Ca2+ efflux (exchange or antiport transport) were very similar.

Animals↗
PubMed2005

Assessment of a 2,4,6-trinitrotoluene-contaminated site using Aporrectodea rosea and Eisenia andrei in mesocosms.

Polynitro-organic compounds such as 2,4,6-trinitrotoluene (TNT) can be released into the environment from production and processing facilities and military firing ranges as well as through field use and disposal practices. Based on laboratory toxicity data, TNT has lethal (at >/=260 mg TNT/kg dry soil) and sublethal effects (at >/=59 mg TNT/kg dry soil) to the earthworm. However, field studies are needed to relate exposure of organisms to explosives in mixed-contaminated soil under field conditions and to define effects-based ecotoxicologic benchmarks for TNT-contaminated soil. In the present study, the lethal and sublethal effects of a 10-day in situ exposure at a TNT-contaminated field site using mesh-bag mesocosms were assessed. In addition to the survival end point, the biomarkers of earthworm exposure and effect-including tissue residues, lysosomal neutral red retention time (NRRT), and total immune activity (TIA)-were measured. Concentrations of TNT in soil mesocosms ranged from 25 to 17,063 mg/kg. Experiments indicated a trend toward decreasing survival of caged Aporrectodea rosea and Eisenia andrei as the concentration of TNT and total nitroaromatic compounds increased. E. andrei tolerated higher concentrations of TNT (up to 4050 mg/kg dry soil) in mesocosms than did indigenous earthworms, who survived only at </=1146 mg TNT/kg. Earthworms E. andrei and A. rose survived in 67% and 75% of TNT-contaminated mesocosms, respectively, compared with references groups. NRRT was significantly decreased in surviving earthworms from the contaminated areas compared with those from the reference site. TIA was not affected by field exposure to TNT. Earthworm tissue concentrations of TNT metabolites 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene were not correlated with TNT soil concentrations. In addition, higher tissue concentrations of TNT metabolites were observed at concentrations ranging from 116 to 130 mg TNT/kg soil. The results showed that earthworm exposure in TNT-contaminated soil produced both lethal and sublethal effects in the field. The results of study indicated that mesocosm experiments would be useful to assess the toxicity of a site and to characterize the overall effects of contaminants. However, mesocosm experiments present special considerations (e.g., abiotic factors, exposure period) when used at heterogenous sites, and data must be interpreted with caution.

Animals↗
PubMed2006

In vitro toxicity evaluation of single walled carbon nanotubes on human A549 lung cells.

This paper describes the in vitro cytotoxicity assessment of single walled carbon nanotubes (SWCNT) on A549 cells, a human lung cell line. Cellular viability was determined using the alamar blue (AB), neutral red (NR) and MTT assays, which evaluated metabolic, lysosomal and mitochondrial activity respectively. In addition, the total protein content of the cells was measured using the coomassie brilliant (CB) blue assay. Supernatants were also assayed for Adenylate Kinase (AK) release and Interleukin 8 (IL-8) which indicated a loss of cell membrane integrity and an inflammation response respectively. To investigate the interactions between serum components in the test medium and the test materials, exposures were conducted both in serum containing (5%) and serum-free medium. Results from the cytotoxicity tests (AB, CB, MTT) revealed the SWCNT to have very low acute toxicity to the A549 cells as all but one of the reported 24h EC(50) values exceeded the top concentration tested (800 microg/ml). The SWCNT were found to interfere with a number of the dyes used in the cytotoxicity assessment and we are currently conducting a comprehensive spectroscopic study to further investigate these interactions. Of the multiple cytotoxicity assays used, the AB assay was found to be the most sensitive and reproducible. Transmission electron microscopy (TEM) studies confirmed that there was no intracellular localization of SWCNT in A549 cells following 24h exposure; however, increased numbers of surfactant storing lamellar bodies were observed in exposed cells.

Adenylate Kinase↗
PubMed2002

Induction and inhibition of aromatase (CYP19) activity by various classes of pesticides in H295R human adrenocortical carcinoma cells.

Various pesticides known or suspected to interfere with steroid hormone function were screened in H295R cells for effects on catalytic activity and mRNA expression of aromatase. Dibutyl-, tributyl-, and triphenyltin chloride decreased aromatase and ethoxyresorufin O-deethylase activities concentration dependently (1-300 nM; 24-h exposure). However, these decreases occurred only at cytotoxic concentrations, indicated by decreases in mitochondrial MTT reduction and intracellular neutral red uptake. The organotins did not cause direct inhibition during the catalytic assay (1-1000 nM; 1.5-h exposure). The same was true for p,p'-DDT, and o,p-DDT, and o,p-DDE, which decreased aromatase activity only at cytotoxic concentrations (> or =10 microM; 24-h exposure). p,p'-DDE had no effect on aromatase activity or cell viability at 1 and 10 microM. Various imidazole-like fungicides were aromatase inhibitors. Imazalil and prochloraz were potent mixed inhibitors (K(i)/K(i)(') = 0.04/0.3 and 0.02/0.3 microM, respectively), whereas propiconazole, difenoconazole, and penconazole were less potent competitive inhibitors (K(i) = 1.9, 4.5, and 4.7 microM, respectively). Fenarimol, tebuconazole, and hexaconazole decreased aromatase activity close to cytotoxic concentrations. Vinclozolin, as was shown previously for atrazine, induced aromatase activity and CYP19 mRNA levels about 2.5- and 1.5-fold, respectively. To investigate the mechanism of aromatase induction in H295R cells, the ability of the pesticides to increase intracellular cAMP levels was examined. Vinclozolin (100 microM) and atrazine (30 microM) increased cAMP levels about 1.5-fold above control. Forskolin and isobutyl methylxanthine (IBMX) increased cAMP levels 3 and 1.8-fold, respectively. Time-response curves for cAMP induction and concentration-response curves for aromatase induction by vinclozolin, atrazine, and IBMX were similar, suggesting that the mechanism of aromatase induction by these pesticides is mediated through inhibition of phosphodiesterase activity.

Aromatase↗
PubMed1996

Cryopreservation of rat and mouse hepatocytes. I. Comparative viability studies.

We have compared the viability of cultures of cryopreserved (CP) rat and mouse hepatocytes with fresh cells with respect to their attachment efficiency, uptake of neutral red (NR), 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carbo xyanilide xyanilide inner salt (XTT) metabolism, and ability to synthesize new proteins. Total recovery of rat hepatocytes after cryopreservation was 42.1 +/- 18.0%, with high viability (82.3 +/- 7.8%, as determined by trypan blue exclusion). These cells had significantly lower attachment efficiencies than fresh cells over 72 hr in culture. Viability of CP rat hepatocyte cultures was lower than fresh cell cultures, but was constant throughout 72 hr (approximately 68%). Total recovery of CP mouse hepatocytes (postthaw viability = 85.4 +/- 6.8%) was 53.6 +/- 14.7%. CP and fresh mouse hepatocyte cultures had similar attachment efficiencies and viabilities. NR uptake by CP rat hepatocyte cultures was significantly higher than in fresh cells at 48 and 72 hr after plating (400% and 810% of fresh cells, respectively). In contrast, CP mouse hepatocytes, which did not detach significantly in culture, took up NR to the same extent as fresh cells. The rate of NR uptake into rat and mouse hepatocytes, cultured for 24 hr, was unaltered by cryo-preservation. XTT metabolism by hepatocytes from either species was not affected by cryopreservation. Protein synthesis over 72 hr, as measured by incorporation of [3H]leucine, was lower in CP cultures than in fresh cells (CP rat hepatocyte protein synthetic activity was 32.3 +/- 6.8% of fresh, and CP mouse hepatocyte protein synthetic activity was 49.0 +/- 10.1% of fresh). Protein synthesis did not alter over 72 hr culture.

Animals↗
PubMed2005

Assessing the detoxication efficiencies of wastewater treatment processes using a battery of bioassays/biomarkers.

A battery of in vitro bioassays, including a Neutral Red (NR) assay using MCF-7 cells for predicting cytotoxic chemicals, an ethoxy resorufin-O-deethylase (EROD) activity assay using H4IIE cells to check for dioxin-like chemicals, and a recombinant gene yeast assay for screening estrogenic chemicals, was conducted to assess the removal efficiencies of trace toxic chemicals by different treatment processes in the waste water treatment plant (WWTP). The effluents were extracted by solid phase extraction (SPE) and were fractionated into three fractions based on polarities. The battery of bioassays was performed for each fraction. In the battery, the toxicities of the effluents were described according to their modes of actions (MOA) or biomarkers and the properties of the toxic chemicals were categorized by their polarities and MOAs. The proposed procedure could be used as a tool to diagnose the toxic characteristics of the complicate mixture. The results showed that cytotoxic, dioxin-like and estrogenic chemicals could be detected in all samples. In the influent, cytotoxic and dioxin-like chemicals were mainly in polar fraction and estrogenic chemicals were in non-polar and moderate-polar fractions. The secondary treatment (active sludge) could remove a small amount of these toxicants. Among different types of advanced treatments, flocculation was good enough to remove most of the cytotoxic chemicals and a combination of flocculation, ozone oxidation, and post-biological treatment could eliminate most of the dioxin-like and estrogenic chemicals.

Animals↗
PubMed1998

In vitro toxicity of zamifenacin (UK-76,654) and metabolites in primary hepatocyte cultures.

1. We compared the sensitivities of primary hepatocytes from rat, dog and monkey to zamifenacin and two major metabolites, the methylenedioxy ring-opened catechol, UK-80,178 and its methylated product, UK-82,201. Toxicity was determined both via neutral red uptake and enzyme leakage data. 2. Canine hepatocytes were most sensitive to the cytotoxic effects of zamifenacin during 24-h exposure. Significant decreases in medium concentrations of zamifenacin in the presence of primary hepatocytes verified cellular uptake during the initial 2-h incubation. All three cell types were much more sensitive to UK-82,201 than to the catechol metabolite or parent drug. 3. The rapid onset of cytotoxicity indicated by elevations of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and other markers in the medium after UK-82,201 exposure, the delayed but substantial cytotoxic response to the parent drug which was suggestive of biotransformation to a reactive moiety, in vivo and in vitro drug metabolism results and subacute toxicology data suggest that dog may more effectively transform zamifenacin into UK-82,201, which is relatively hepatotoxic. 4. Because the catechol was generally less toxic than the O-methylated product, species that eliminate zamifenacin primarily as the catechol or its conjugate may be less affected by the potential hepatotoxicity of the methylated product. Our studies show that dog is the most sensitive species due to metabolism of the common catechol metabolite. The low incidence of potential hepatotoxicity in the clinic points to rare but important differences in the metabolism of Zamifencin. We conclude that the findings in dog were not predictive of subsequent effects in man.

Alanine Transaminase↗
PubMed1995

Toxic effects of anabolic-androgenic steroids in primary rat hepatic cell cultures.

Hepatic complications in athletes and bodybuilders after abusing anabolic-androgenic steroids (AAS) have been reported. Hepatic injury, including cholestasis, peliosis hepatis, hyperplasia, and tumors, have been attributed to abuse of the 17 alpha-alkylated AAS. Some of these pathological conditions have been reversed when individuals were converted to nonalkylated AAS regimens. The purpose of this study was to determine and compare the direct toxic effects of commonly abused AAS (both 17 alpha-alkylated and nonalkylated) in primary hepatic cell cultures. Primary cultures, established from 60-day-old Sprague-Dawley rats, were exposed to doses of 1 x 10(-8), 1 x 10(-6), and 1 x 10(-4)M 19-nortestosterone, fluoxymesterone, testosterone cypionate, stanozolol, danazol, oxymetholone, testosterone, estradiol, and methyltestosterone for 1, 4, and 24 hr. Lactate dehydrogenase (LDH) release, neutral red (NR) retention, and glutathione (GSH) depletion were evaluated to determine plasma membrane damage, cell viability, and possible oxidative injury, respectively. Those cultures exposed to the 17 alpha-alkylated AAS, methyltestosterone and stanozolol, at doses of 1 x 10(-4) M for 24 hr and the 17 alpha-alkylated AAS, oxymetholone, at 1 x 10(-4) M for 4 and 24 hr showed significant increased in LDH release and decreases in NR retention while there were no significant differences with the nonalkylated steroids (testosterone cypionate, 19-nortestosterone, testosterone, and estradiol). GSH depletion was evaluated in cultures treated with 1 x 10(-8), 1 x 10(-6), and 1 x 10(-4) M concentrations of methyltestosterone, stanozolol, and oxymetholone for 1, 2, 4, and 6 hr. Cultures exposed to 1 x 10(-4) M oxymetholone were significantly depleted of GSH at 2, 4, and 6 hr; cultures exposed to 1 x 10(-4) M methyltestosterone were significantly depleted of GSH at 4 and 6 hr; and cultures exposed to stanozolol were not significantly depleted of GSH at any of the time periods tested. These data indicate that the 17 alpha-alkylated steroids (methyltestosterone, oxymetholone, and stanozolol) are directly toxic to hepatocytes, whereas the nonalkylated steroids (testosterone cypionate, 19-nortestosterone, testosterone, and estradiol) show no effects at similar doses. These data demonstrate a trend toward a structural-activity relationship to AAS-induced toxicity in primary cultures of rat hepatocytes.

Anabolic Agents↗
PubMed1997

Construction of mutants of Salmonella typhimurium deficient in 8-hydroxyguanine DNA glycosylase and their sensitivities to oxidative mutagens and nitro compounds.

8-Hydroxyguanine (8-OH-G) DNA glycosylase is an enzyme involved in repair of oxidative DNA damage, e.g., 8-OH-G in DNA. In order to assess the roles of 8-OH-G in spontaneous and chemically-induced mutagenesis, the mutMST gene encoding 8-OH-G DNA glycosylase of Salmonella typhimurium was disrupted in several Ames tester strains, i.e., S. typhimurium TA1535 (hisG46, uvrB-, rfa), TA1975 (hisG46, uvr+, rfa) and TA102 (hisG428, uvr+, rfa). The spontaneous mutation frequencies were increased 2.4 and 1.6 times, respectively, by the mutMST deletions in strains TA1535 and TA1975, which are spontaneously reverted to His+ by mutations mainly at G:C base pairs. The resulting strains YG3001 (TA1535 delta mutMST) and YG3002 (TA1975 delta mutMST) were 2 to 8 times more sensitive to the mutagenicities of methylene blue plus visible light, neutral red plus visible light and 2-nitrofluorene than the parent strains. The strain YG3002 but not YG3001 was about 30 times more sensitive to the mutagenicity of 4-nitroquinoline N-oxide than the parent strain TA1975. Neither hydrogen peroxide nor phenazine methosulfate was mutagenic in the mutMST-deletion strains as well as in the parent strains. In contrast, the mutMST deletion did not affect the spontaneous mutation frequency of strain TA102, which has an A:T base pair at the critical site for reversion. The sensitivities of strain TA102 to the chemicals were not enhanced by the mutMST deletion except for hydrogen peroxide. These results suggest that 8-OH-G in DNA plays important roles in spontaneous mutagenesis occurring at G:C base pairs in S. typhimurium, and some nitro aromatics such as 4-nitroquinoline N-oxide or 2-nitrofluorene as well as the photosensitizers plus visible light can produce 8-OH-G in DNA, thereby inducing mutations. In the case of 4-nitroquinoline N-oxide, 8-OH-G rather than DNA adducts seems to play major roles in mutagenesis in uvr+ background. The new strains could be useful for the evaluation of the roles of 8-OH-G in mutagenesis in S. typhimurium and permit the efficient detection of some oxidative mutagens in the environment.

4-Nitroquinoline-1-oxide↗