In vitro metabolism and toxicity assessment of toxin T-514 (Peroxisomicine A1) of Karwinskia humboldtiana in microsomes and primary cultured hepatocytes.

T-514 (Peroxisomicine A(1)) from Karwinskia humboldtiana is a dimeric hydroxyanthracenone with a highly selective cytotoxic effect on tumor cells. We evaluated the metabolism of this compound in two in vitro systems (liver microsomes and hepatocytes) and assessed the cytotoxicity of its metabolites on normal and tumor cells. Microsomes (12.5, 125 and 250 microg of protein/ml) and hepatocytes (1 x 10(6) cells/ml) were incubated with the toxin (25 microM) for 0.5, 1, 3, 6, 9, 12 and 24 h and the samples were examined using chromatographic analysis and UV spectra. Two metabolites (M1 and M2) were detected in the rat microsomes and one (M1) in the monkey microsomes. The retention times and UV spectra of the peaks were very similar to those of the toxin T-514. M1 was isolated and identified as a mixture of two isomers. The cytotoxicity of the metabolites was evaluated in Chang liver and Hep G2 cells but they did not show the selective cytotoxic effect on tumor cells seen in the original compound.

Effect of peroxisomicine A2 and T 544 of the genus Karwinskia on peroxisomes of Candida boidinii.

Dimeric anthracenones have been isolated from toxic plants of the genus Karwinskia (Rhamnaceae). T 514 or peroxisomicine A1 is one of the anthracenonic compounds which produce irreversible and selective damage on the peroxisomes of yeast cells in vivo. In this paper we describe the effect of two structurally related anthracenones on cell viability and on the peroxisomes of the methylotrophic yeast Candida boidinii. As has been described for peroxisomicine A1, peroxisomicine A2 and T 544 caused a decrease in the viability of C. boidinii at all concentrations tested, and disruption of the peroxisomal membrane, T 544 showing the strongest effect. In C. boidinii cell death and peroxisomal damage seem to be independent events.

Effect of toxins 544 and 514 from Karwinskia humboldtiana (buckthorn) plant upon fetal development of the mouse.

The teratogenic effect of toxins 544 and 514 from K. humboldtiana upon the mouse embryo was evaluated. One half of the LD50 dose for the mouse was administered at day 8 of gestation. At the end of pregnancy, reproduction and fetal data were recorded. Dams treated with toxin 544, but not with toxin 514, showed a higher incidence of reabsorptions, malformations, as well as lower fetal length compared to the control group.

Studies on the effect of peroxisomicine on catalase activity in albino mice.

Peroxisomicine is a toxic compound isolated from plants of the genus Karwinskia (Rhamnaceae). This toxin produces irreversible and selective damage to the peroxisomes of yeast cells in vivo. Peroxisomicine also inhibits catalase activity in vitro, when using purified enzyme. This paper reports on the effect of peroxisomicine on liver catalase in tissue fragments, in situ, as well as in mice intoxicated with peroxisomicine, in vivo. The catalase activity was determined by biochemical and histochemical methods. In contrast with the reported findings in vitro, the results demonstrate that there is no inhibition of the activity of tissue catalase, and suggest that catalase in situ and in vivo is protected against the inhibitory effect of peroxisomicine by an unknown factor.

Comparison of the hepatotoxicity of toxin T-514 of Karwinskia humboldtiana and its diastereoisomer in primary liver cell cultures.

Toxin T-514 of Karwinskia humboldtiana has been demonstrated to be hepatotoxic in vivo and in vitro. Recently a diastereoisomer of T-514 has been isolated. In the present study we have evaluated and compared the in vitro hepatoxicity of the diastereoisomer of T-514 using primary cultures of rat hepatocytes. Cytotoxicity was evaluated by release of cytoplasmic enzyme lactate dehydrogenase (LDH), and mitochondrial metabolic function (MTT reduction). The diastereoisomer was shown to be almost as hepatoxic in vitro as toxin T-514.

Peroxisomicine A1 (plant toxin-514) affects normal peroxisome assembly in the yeast Hansenula polymorpha.

Previously we demonstrated that peroxisomicine A1 (T-514), a plant toxin isolated from Karwinskia species, has a deteriorating effect on the integrity of peroxisomes of methylotrophic yeasts. Here we describe two strains of Hansenula polymorpha, affected in the normal utilization of methanol as sole source of carbon and energy due to peroxisomicine A1 treatment. The two strains isolated (L17 and RV31) grew poorly on methanol, apparently due to malfunctioning of their peroxisomes. Moreover, the cells displayed a high peroxisome turnover rate. We argue that the peroxisomicine A1 induced phenotype of both strains is due to a genomic mutation. Strain L17 was functionally complemented after transformation with a H. polymorpha genomic library. The complementing 2.8 kb DNA fragment did not contain a well-defined ORF and led us to speculate that it may contain regulatory sequences that, when present in multiple copies in the cell, result in a change of expression of specific genes, thus causing restoration of normal methylotrophic growth.

Effects of toxin T-544 from the Karwinskia humboldtiana (buckthorn) plant upon mouse embryos explanted at 11 days.

Eleven-day mouse embryos were exposed to the K. humboldtiana toxin T-544 for 24 hr. At the end of the culture period, embryos were examined grossly for malformations and biochemically for altered protein levels. There was a significant difference in malformations in those embryos exposed to 0.05 and 0.2 microgram/ml of toxin compared with controls. Embryo protein content was significantly lower in those embryos exposed to 0.1 microgram/ml of T-544 compared with control group.

In vitro selective toxicity of toxin T-514 from Karwinskia humboldtiana (buckthorn) plant on various human tumor cell lines.

Toxin T-514 is a dimeric anthracenone isolated from the Karwinskia humboldtiana (buckthorn) plant. Its potential anti-neoplastic effect was evaluated in vitro and the results obtained were compared with the effect of other known anti-cancer agents. Normal and malignant continuous cell lines were tested. After a 72-h exposure, neoplastic cells derived from hepatic, pulmonary and colonic tissues were more sensitive to toxin T-514 than normal cells from the corresponding organ. Hepatoma cells and colon adenocarcinoma CT50 values were < 10 micrograms/ml. Lung adenocarcinoma, undifferentiated bronchogenic cancer cells and small cell carcinoma CT50 values were < 20 micrograms/ml. All benign cell CT50 levels tested were > 113 micrograms/ml. This in vitro selective toxicity found with toxin T-514 was also seen with 5-fluororacil and mitomycin for colon adenocarcinoma and with epidoxorubicin for undifferentiated bronchogenic cancer cells.

Toxicity assessment of toxins T-514 and T-544 of buckthorn (Karwinskia humboldtiana) in primary skin and liver cell cultures.

The present study was undertaken to assess and compare the in vitro cytotoxicity of toxins T-514 and T-544 of Buckthorn (Karvinskia humboldtiana) using primary cultures of rat hepatocytes and keratinocytes. Cell cultures were exposed to 6, 12, 25 and 50 microM concentrations of the toxins for 2, 4, 6 and 24-h periods. Cytotoxicity was determined by release of the cytoplasmic enzyme, lactate dehydrogenase (LDH), in culture media, methylthiazoltetrazolium (MTT) reduction and neutral red (NR) uptake. An increase in LDH leakage was observed in liver cell cultures as early as 2 h with 50 microM T-544 and with 6 microM T-514 and T-544 at 6 h and 24 h, respectively. In the NR assay the toxicity was evident at 2 h with 12 microM T-514 and T-544 and with 6 microM concentrations of both toxins at 6 h. On the other hand, a decrease in MTT reduction was detected at 4 h with 50 microM concentrations of both toxins and with 25 microM T-544 and 12 microM T-514 at 6 h and 6 microM T-514 and T-544 at 24 h. Both toxins were shown to be highly hepatotoxic; T-514 was more toxic than T-544. In the skin cell cultures, the toxicity of the toxins was not as severe and was not expressed until 12 h of exposure.

Toxicity assessment of toxins T-514 and T-544 of buckthorn (Karwinskia humboldtiana) in primary skin and liver cell cultures.

The present study was undertaken to assess and compare the in vitro cytotoxicity of toxins T-514 and T-544 of buckthorn (Karwinskia humboldtiana) using primary cultures of rat hepatocytes and keratinocytes. Cell cultures were exposed to 6, 12, 25 and 50 microM toxins for 2-, 4-, 6- and 24-h periods. Cytotoxicity was determined by release of the cytoplasmic enzyme, lactate dehydrogenase (LDH), in culture media, methylthiazoltetrazolium (MTT) reduction and neutral red (NR) uptake. An increase in LDH leakage was observed in liver cell cultures as early as 2 h with 50 microM T-544 and with 6 microM T-514 and T-544 at 6 h and 24 h, respectively. In the NR assay the toxicity was evident at 2 h with 12 microM T-514 and T-544 and with 6 microM concentrations of both toxins at 6 h. On the other hand, a decrease in MTT reduction was detected at 4 h with 50 microM concentrations of both toxins and with 25 microM T-544 and 12 microM T-514 at 6 h and 6 microM T-514 and T-544 at 24 h. Both toxins were shown to be highly hepatotoxic; T-514 was more toxic than T-544. In the skin cell cultures, the toxicity of the toxins was not as severe and was not expressed until 12 h of exposure.

Optimization and validation of an analytical procedure by high-performance liquid chromatography for the quantification of peroxisomicines and isoperoxisomicines.

Peroxisomicine A1 is a potential antineoplastic substance extracted from plants of the genus Karwinskia. An RP-HPLC-DAD method was developed and validated for the separation and quantification of four isomers of this compound. These isomers coelute in the preparative procedure and are present at a proportion ranging between 3 and 5% in the peroxisomicine A1 purified in the laboratory. The desirability coefficient of the method described here was enhanced 140% with respect to the previously reported method.

Experimental evidence for toxic damage induced by a dimeric anthracenone: diast T-514 (peroxisomicine A2).

Dimeric anthracenones obtained from the genus Karwinskia (Rhamnaceae) are characteristic compounds isolated from the plants of this species. Previous toxicity studies demonstrated Diast T-514 to be toxic to animals in experimental settings. Diast T-514 extracted and characterized from Karwinskia parvifolia, was studied in CD1 mice. The LD50 for this compound was determined. Animals were tested with Diast T-514 following enteral and parenteral administration. An LD50 dose by both oral and intraperitoneal administration showed selective damage to target organs.

Quantitative analysis of liver peroxisomes in rats intoxicated with peroxisomicine-A1.

Peroxisomes are single-membrane-bound organelles present in almost all eukaryotic cells. Hypolipidemic agents such as clofibrate, herbicides and plasticizers induce an increase in the number and size of peroxisomes from mammalian cells. However, there is no evidence of drugs causing a decrease in the number of these organelles. In this paper, we report the effect in vivo of toxin T-514 extracted from the plant Karwinskia humboldtiana, now re-named peroxisomicine-A1, on hepatic peroxisomes from rats intoxicated with this compound. Rats were treated with a single dose of 25 mg/kg of peroxisomicine-A1 and at different times were killed by decapitation. For the peroxisomal counting, liver tissue sections from control and treated rats were processed for the localization of catalase in peroxisomes. The results of the quantitative analysis demonstrated a significant decrease in the number of liver peroxisomes from rats intoxicated with peroxisomicine-A1. This finding suggests that peroxisomicine-A1 as in yeast, causes a damage to mammalian peroxisomes. The diminution in the number of peroxisomes could be a consequence of damage to the organelle, which is further removed by an autophagic process.

Production of reactive oxygen species by toxin T-514 of genus Karwinskia in vitro.

In the present study we have analyzed the production of reactive oxygen species by toxin T-514 of the genus Karwinskia in vitro (primary liver cell cultures and microsomes), as well as their possible role in its cytotoxicity. The role of catalase and superoxide dismutase (SOD) as defense mechanisms against oxidative stress was also studied. Freshly isolated hepatocytes or microsomes were exposed to T-514 in the presence or absence of catalase and SOD. Cytotoxicity was determined by methylthiazoltetrazolium (MTT) reduction. Oxidative stress was evaluated by the dichlorofluorescein diacetate (DCFDA) fluorescent probe and the reduction of ferricytochrome c. Exposure of hepatocytes to toxin T-514 for 2-, 4-, 6- and 24-h periods resulted in a time- and concentration-dependent increase in the suppression of mitochondrial metabolic activity. T-514 induced the production of reactive oxygen species in both hepatocytes and microsomes. Catalase and superoxide dismutase had a protective effect against the cytotoxicity of T-514 in hepatocytes and also inhibited the production of oxygen reactive species in microsomes. The results indicate that oxidative stress mediated by reactive intermediates may be a mechanism by which T-514 induces its cytotoxic effect.

In vitro binding studies of the peroxisomicine A1-BSA and -HSA interactions.

Peroxisomicine A1 (PA1) is a dimeric hydroxyanthracenone isolated from fruits of plants belonging to the genus Karwinskia. Showing selective toxicity between malignant and benign cell lines, it is currently under screening as an antineoplastic agent. Very little is known about its mechanism of action. In the present work the extent of binding of this substance with Bovine Serum Albumin (BSA) and Human Serum Albumin (HSA) at pH 7.2 and 7.4 has been evaluated using the spectrophotometric method. Absorbance of PA1 was altered by the presence of albumin and this property was used to generate binding isotherms. The investigation was carried out at four different temperatures. The data were analyzed by assuming two types of binding sites. Results indicated that PA1 binds to both albumins at physiological pH, which is reflected by the affinity constants of the order of 10(5). There are two types of binding sites in the albumin for PA1; with the electrostatic forces being discarded, the hydrophobic and hydrogen bond are more probable. Binding with HSA is stronger than with BSA.

Effect of meso-2,3-dimercaptosuccinic acid on urinary lead excretion in exposed men.

The efficacy of meso-2,3-dimercaptosuccinic acid (DMSA) was evaluated in workers occupationally exposed to lead (Pb; blood level >50 microg/dL). Ten men were given 600 mg of DMSA per orem daily, for five days. Pb concentrations of whole blood and urine were determined throughout therapy. Hematology analyses, blood chemistry, and urinalysis were obtained at the start of the study, at the end of the DMSA treatment, and at 72 hours after the administration of the final dose. DMSA therapy had no influence on hepatic, hematologic, or renal functions and was effective in decreasing the concentration of blood Pb in all the subjects without adverse drug reactions.

[Familial poisoning with Karwinskia humboldtiana]. / Intoxicación de una familia por Karwinskia humboldtiana (tullidora).

The ingestion of ripe fruit of the Karwinskia humboldtiana, a shrub commonly known as tullidora or coyotillo, produces an intoxication described in the literature as a symmetric flaccid paralysis of the hind limbs, progressive and ascendent, that in severe cases may cause bulbar paralysis and death. The cause of an acute accidental intoxication of an entire family is presented here, wherein ten out of thirteen members ingested the ripe fruit of the tullidora. Three died, the father and two daughters. For the first time the toxins determination in blood by thin layer chromatography method is described. This method supports the diagnosis with other polyradiculoneuritis such as poliomyelitis and the Guillain Barre's syndrome.

Evaluation of urinary mercury excretion after administration of 2,3-dimercapto-1-propane sulfonic acid to occupationally exposed men.

The purpose of this study was to determine the clinical efficacy of 2,3-dimercapto-1-propane sulfonic acid, Na salt, on the urinary excretion of mercury as well as its possible adverse effects. Ten men with occupational mercury exposure (urinary level of 50 micrograms/g creatinine or more) were assigned to receive 2,3-dimercapto-1-propane sulfonic acid p.o. (DIMAVAL capsules, 100 mg) 300 mg/d for five days. Informed written consent was obtained from each subject. Hematology analyses, blood, chemistry, and urinalysis were obtained at the start of the study, at the end of the 2,3-dimercapto-1-propane sulfonic acid treatment and 72 hours after the administration of the final dose of 2,3-dimercapto-1-propane sulfonic acid. Twenty-four-hour urine mercury levels were closely monitored throughout therapy. All data and measurements before and during drug doses were evaluated by analyses of variance. In all subjects mean urine mercury was significantly increased (p < .05) after pre-2,3-dimercapto-1-propane sulfonic acid treatment. One subject had a moderate hypersensitivity reaction (rash) to 2,3-dimercapto-1-propane sulfonic acid but no other toxic effects were observed.