Optimisation of cell-based assays for medium throughput screening of oxidative stress.

Identification and development of cell-based assays adapted to medium throughput screening requirements is important when screening chemicals for their potential toxic properties. We describe here rapid fluorometer-based and spectrophotometer-based microplate assays which allow for the evaluation of oxidative stress in hepatocyte cell cultures by measurement of three markers: production of hydroperoxide assessed by the DCFH-DA probe, cellular antioxidant status by measurement of reduced glutathione and glutathione peroxidase activity and cytotoxicity and mitochondrial damage by evaluation of the mitochondrial transmembrane potential with rhodamine 123 fluorescent dye. The assays described here are rapid, simple and inexpensive, which are desirable when setting up screening assays. This system should be useful in selecting candidate compounds during the pre-development phase of agrochemicals or pharmaceuticals. It should also be of interest for retrospective and explanatory studies of mechanisms underlying toxicity observed in vivo.

In vitro toxicity of trichothecenes on human erythroblastic progenitors.

Trichothecenes are mycotoxins produced by various species of fungi which can occur on various agricultural products. Among these compounds, T-2 toxin, HT-2 toxin, diacetoxyscirpenol (DAS) and deoxynivalenol (DON) are the most naturally encountered and potent trichothecenes. Consumption of trichothecene contaminated foods by farm animals and humans leads to mycotoxicoses. Trichothecenes are known to induce haematologic disorders such as neutropenia, thrombopenia, and aplastic anemia in human and animals. The aim of our investigations is to explore the effects of trichothecenes on the haematopoietic progenitors. The four trichothecenes previously demonstrated to be strongly cytotoxic for human CFU-GM have been tested on human BFU-E. For this purpose, a culture model of human erythroblastic progenitors (BFU-E) optimized for toxicological studies was used to determine the effects of T-2, HT-2, diacetoxyscirpenol (DAS) and deoxynivalenol (DON) on red blood cell precursor proliferation and differentiation. Results showed that human BFU-E are as sensitive to trichothecenes as human CFU-GM, except for DON, in the range of concentrations tested. Differentiation of erythroblastic progenitors could be perturbed by these mycotoxins. Human erythroblastic progenitors are also a target of trichothecenes.

Comparison of toxicity induced by T-2 toxin on human and rat granulo-monocytic progenitors with an in vitro model.

T-2 toxin is a trichothecene mycotoxin produced by various species of fungi. Trichothecenes are known as major contaminants of cereals and their derivatives. In man as well as in animals, T-2 toxin has been shown to induce alimentary intoxication and, among others, haematological symptoms. Granulo-monocytic progenitors from human umbilical cord blood on the one hand and granulo-monocytic progenitors from rat bone marrow on the other, were cultured in the presence of T-2 toxin (from 10(-7) to 10(-10) M) for 14 days. A study of concentration and effect relationships showed a strong and rapid effect of T-2 toxin on rat colony forming unit-granulocyte and macrophage (CFU-GM) between 5 x 10(-9) M and 10(-9) M. On the other hand, human CFU-GM were able to grow in the presence of the same T-2 toxin concentrations. IC50 were determined on day 7, 10 and 14. They were, respectively, 1.6 x 10(-9) M; 3.6 x 10(-9) M; 1.4 x 10(-9) M for human cells, and 2.2 x 10(-9) M; 3.3 x 10(-9) M; 2.6 x 10(-9) M for rat cells. The present study was prompted by the need to define precisely the cytotoxic and inhibitory T-2 toxin concentrations for rat and human CFU-GM. It is particularly relevant for the investigation of cellular T-2 toxin targets and in order to elucidate the mechanism of trichothecene haematotoxicity.

Comparison of toxicity induced by HT-2 toxin on human and rat granulo-monocytic progenitors with an in vitro model.

HT-2 toxin is a trichothecene mycotoxin occurring naturally in various agricultural products. Many in vitro studies have shown that HT-2 toxin is a major metabolite of the parent compound T-2 toxin. In man as well as in animals both toxins have been shown to cause alimentary intoxications and haematological disorders. Granulomonocytic progenitors (CFU-GM) from human umbilical cord blood and from rat bone marrow were cultured in the presence of HT-2 toxin (10(-7) M to 10(-10) M) for 14 days. The concentration-effect relationship was studied and the IC50 were determined on Days 7, 10 and 14. The IC50 was 1.8 x 10(-9) M, 3.5 x 10(-9) M and 1.8 x 10(-9) M for human cells, and 2 x 10(-9) M, 2.3 x 10(-9) M and 2.2 x 10(-9) M for rat cells. The results have been compared with previous findings for T-2 toxin. Although T-2 and HT-2 toxins had a similar IC50 on human and rat CFU-GM, the expression of their cytotoxicities was different. These findings are relevant to investigation of the cellular targets of T-2 and HT-2 in elucidating the mechanism of trichothecene haematotoxicity.

Effects of a diphenyl-ether herbicide, oxyfluorfen, on human BFU-E/CFU-E development and haemoglobin synthesis.

The diphenyl-ether herbicides exert their phytotoxic activity by preventing chlorophyll formation in plants as a result of inhibition of protoporphyrinogen oxidase. This enzyme is the last step of the common pathway for chlorophyll and haem biosynthesis. The aim of this work is to determine whether herbicide inhibitors of plant protoporphyrinogen oxidase could act on the human protoporphyrinogen oxidase involved in haemoglobin synthesis and cause heamatologic diseases. Human erythroblastic progenitors (BFU-E/CFU-E: Burst Forming Unit-Erythroid and Colony Forming Unit-Erythroid) were exposed to oxyfluorfen, a diphenyl-ether herbicide in the presence of erythropoietin, and the haematoxicity evaluated in vitro by scoring the development of BFU-E/CFU-E colonies after 7 and 14 days of culture. The toxic effect on differentiation has been evaluated using four criteria: morphology, total protein, total porphyrin, and haemoglobin content. The study of BFU-E/CFU-E proliferation and differentiation showed a cytotoxic effect of oxyfluorfen only at very high concentrations. In contrast, haemoglobin synthesis can be inhibited by concentration of oxyfluorfen (10(-4) M) that have no adverse effect on cellular proliferation.

In vitro toxicity induced by deoxynivalenol (DON) on human and rat granulomonocytic progenitors.

Deoxynivalenol (DON) is a trichothecene mycotoxin produced by various species of fungi. Trichothecenes are known as major contaminants of cereals and cereal-containing foods. DON has been detected in agricultural products worldwide and persists in products after processing. In humans as well as in animals, DON has been shown to induce both alimentary and hematological toxicities. Granulo-monocytic progenitors (CFU-GM) from human umbilical cord blood from rat bone marrow were cultured in the presence of DON (from 10(-6) to 10(-8) mol/L) for 14 days. DON rapidly inhibits human and rat CFU-GM in a concentration-dependent manner between 10(-6) and 2.5 x 10(-7) mol/L. IC50 values on days 7, 10, and 14 were, respectively, 3 x 10(-8), 2.9 x 10(-8), 3.9 x 10(-8) mol/L for human CFU-GM and 2.6 x 10(-7), 1.5 x 10(-7), and 1.6 x 10(-7) mol/L for rat CFU-GM. The present study defines the cytotoxic and inhibitory DON concentrations for rat and human CFU-GM and provides a system for further investigation of cellular DON targets and elucidation of the mechanism of trichothecene hematotoxicity. Moreover, we propose one of the trichothecenes tested in our studies as a reference molecule for in vitro studies, since one mycotoxin seems to be the most potent myelotoxic inhibitor of CFU-GM detected to date.

In vitro effects of diacetoxyscirpenol (DAS) on human and rat granulo-monocytic progenitors.

Diacetoxyscirpenol (DAS) is a trichothecene mycotoxin produced by various species of fungi. Trichothecenes are known as major contaminants of cereals and cereal-containing foods. DAS has been detected in agricultural products worldwide and persists in products after processing. In human as well as in animals, DAS consumption has been shown to induce haematological disorders (neutropenia, aplastic anemia). Granulo-monocytic progenitors (CFU-GM) from human umbilical cord blood and rat bone marrow have been cultured in the presence of DAS (from 10(-8) M to 5 x 10(-10) M) for 14 days. Study of concentration and effect relationships have shown a sharp effect of DAS on rat CFU-GM between 10(-7) M and 10(-8) M, while human CFU-GM are able to grow in the presence of 10(-8) M of the toxin. IC50 values on day 14 are respectively, 7.6 x 10(-9) M for human CFU-GM and 6.2 x 10(-9) M for rat CFU-GM.

Flavonoids inhibit genetic toxicity produced by carcinogens in cells expressing CYP1A2 and CYP1A1.

The effects of the flavonoids quercetin, apigenin and chrysin (10 microM) on the genetic toxicity of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and benzo[a]pyrene (BaP) was investigated at sub-cytotoxic concentrations in Chinese hamster V79 cells expressing human or rat cytochromes P450. In V79 r1A2-NH and V79 h1A1-MZ cells, none of the flavonoids increased DNA strand breaks (SB) (measured by the Comet assay) or produced detectable DNA adducts (measured by 32P-post-labelling). Neither IQ nor BaP produced DNA damage in the absence of expressed CYP1A2 or CYP1A1, respectively. DNA damage measured as SB and DNA adducts was detectable in V79 r1A2-NH cells expressing rat CYP1A2 when treated with IQ (2.5-50 microM) and this was inhibited by quercetin. Likewise, DNA damage (SB and DNA adducts) was elevated in V79 h1A1-MZ cells expressing human CYP1A1 when treated with BaP (0.1-0.5 microM) and this was inhibited by chrysin and apigenin, but not by quercetin. The specificity of CYP1A1 inhibition by chrysin and apigenin and CYP1A2 inhibition by quercetin was confirmed by ethylresorufin O-deethylase assay.