Dietary cancer risk from conditional cancerogens (tumor promoters) in produce of livestock fed on species of spurge (Euphorbiaceae). V. Skin irriitant and tumor-promoting diterpene ester toxins of the tigliane and ingenane type in the herbs Euphorbia nubica and Euphorbia helioscopia contaminating fodder of livestock.

Irritant diterpene ester toxins were isolated from Euphorbia nubica and E. helioscopia, which are contaminants of the green fodder of livestock in Egypt. Fractionations of methanol extracts of aerial parts of both plants were monitored by the irritation unit on the mouse ear. Plant extracts were subjected to multiplicative distribution methods, yielding irritant hydrophilic fractions that were further purified by column chromatography. Final purification of the materials was achieved by TLC (silica gel) followed by HPLC, or by TLC alone. In this way, from E. nubica, five Euphorbia factors (Nu1-Nu5) were isolated and characterized as short-chain polyfunctional diterpene esters of tigliane-type parent alcohols. The two weak irritants Nul and Nu3 were triesters of 4-deoxy(4alpha)phorbol. Nu2 was shown to be a triester of the stereoisomeric tigliane-type parent alcohol 4-deoxyphorbol. Weak irritant Nu4 probably is a positional isomer of Nu2. Nu5 was characterized as a short-chain triester of 4,20-dideoxy-5xi-hydroxyphorbol. From E. helioscopia, six short- to medium-chain polyfunctional diterpene esters of the ingenane type, generally containing unsaturated acids were obtained, i.e., four irritant esters of ingenol (Euphorbia factors H1, H2, H5, and H6) and two esters of 20-deoxyingenol (non-irritant Euphorbia substance HS4, and irritant Euphorbia factor H8). All irritant Euphorbia factors of the tigliane and ingenane diterpene ester type described in this investigation are considered to be more or less active tumor promoters, i.e., conditional (non-genotoxic) cancerogens. The Euphorbia factors assayed exhibited moderate (H1) to low (H8) relative tumor-promoting potency in comparison to the ingenane prototype DTE tumor promoter 3-TI.

Dietary cancer risk conditional cancerogens in produce of livestock fed on species of spurge (Euphorbiaceae). I. Skin irritant and tumor-promoting ingenane-type diterpene esters in E. peplus, one of several herbaceous Euphorbia species contaminating fodder of livestock.

The hypothesis was proposed that there is a risk of dietary cancer from conditional cancerogens in produce of livestock polluted with irritants of the diterpene ester type, picked up by feeding on species of Euphorbiaceae (spurge). To challenge this, several herbaceous plants of the genus Euphorbia, widespread as weeds and contaminants of livestock fodder, were identified botanically and extracts of their aerial parts were tested for irritancy on the mouse ear. As compared to a standard probe of croton oil, the extracts of E. peplus, E. nubica and E. helioscopia displayed irritancy. The most active extract (that from E. peplus) was investigated by a fractionation procedure monitored by the mouse ear assay, and five molecularly uniform irritant Euphorbia factors Pe1-Pe5 were identified as diterpene ester-type toxins. Together these factors comprise at least 11 ppm in the aerial parts. They were characterized individually to carry the diterpene parent alcohols ingenol, 20-deoxyingenol, and 20-deoxyingenol-6 alpha, 7alpha-epoxide. The irritancy of the aerial plant parts was shown to be caused mainly by the Euphorbia factors Pe1 and Pe2 together. Upon chronic administration of these irritants and hyperplasiogens as principal cancerogenic risk factors in the mouse skin initiation/promotion bioassay, Pe1 and Pe2 were established as tumor promoters. These findings together support the initial hypothesis and suggest the need for further investigations to determine whether there is a consequent risk of dietary cancer.

Bioavailability to rats and toxicological potential in mice of bound residues of malathion in beans.

Under conditions of local practice, the application of 2,3-succinate-14C-malathion on beans led to the formation of 17-18% of bound 14C-residues after 30 weeks. When fed to rats, 75% of these residues became bioavailable after 2 days with the major part, excreted via expired air (8%) and urine (60%). The main radioactive metabolites detected in urine were malathion monocarboxylic acid and malathion dicarboxylic acid. Feeding of bound residues to mice (1.8 ppm in feed) for 90 days resulted in a reduction in body weight gain after 60 days and inhibition of erythrocyte cholinesterase activity after 90 days. Increased levels of serum glutamic oxaloacetic transaminase and alkaline phosphatase were also observed. The results strongly suggest that bean-bound malathion residues can cause adverse biological effects in mice.

Bioavailability to rats and toxicity in mice of carbofuran residues bound to faba beans.

14C-carbofuran penetrated readily into seeds of Vicia faba and the rate of penetration was found to be dose dependent. The percentage of bound residues was generally low and did not exceed 3% of the applied dose. When the bound residues were fed to rats 46% of the radioactivity was eliminated via CO2 and urine, while tissues contained 25%. Carbofuran phenol and 3-hydroxy carbofuran represented the main metabolites in the urine. These data indicate that bean-bound carbofuran residues are highly bioavailable to rats. Feeding mice with bound carbofuran residues for 90 days led to inhibition of erythrocyte cholinesterase activity after 30 days (35-40%) while the plasma enzyme remained unaffected. Serum transaminases and blood urea nitrogen were significantly elevated, indicating injury to hepatic and renal structures. The results strongly suggest that the bound residues can induce adverse biological effects in mice.

Bioavailability to rats and toxicity of bound residues in bean seeds fumigated with 14C-methyl bromide.

Fumigation of Vicia faba seeds with 14C-methyl bromide for two weeks followed by four-week aeriation period resulted in the formation of bound residues equivalent to 33% of the terminal residue or approx. 30 ppm. Of this amount, 3.4% was incorporated into the seed DNA. The effect of cooking on the treated-aerated beans showed that 50% of the initial dose remained bound to seed tissues. A bioavailability study of 14C-bound residues showed that over 70% of the ingested dose was bioavailable to the rat. The major radioactivity was eliminated in urine (36.6%), feces (24.1%) and CO2 (19.3%). Analysis of the urine showed that 36% of the radioactivity was in the purine fraction; one fifth of this amount was associated with 7-14C-methylguanine. Subacute feeding of bound residues to mice for 8 weeks at a dietary level of 30 ppm resulted in a significant decrease in the number of white blood cells and an increase in the activity of SGOT and SGPT of the treated mice. Blood urea nitrogen was also increased while blood creatinine remained unaltered. The data indicate that seed-bound 14C-methyl bromide residues are highly bioavailable to the rat. The bound residue also possesses a toxicological potential, as manifested by hepatic and glomerular injuries in mice.

Investigations on trifluralin binding to soil and possible uptake of bound residues by plants.

3H-labelled trifluralin was synthesized by condensation of 3H-4-chloro-3,5-dinitro-alpha,alpha,alpha-triflurotoluene with di-n-propyl-amine. The labelled herbicide was applied to autoclaved and non-autoclaved heavy, light and sandy soils at 30 ppm. The treated samples were incubated for 2 months, then extracted and analyzed for metabolic products and bound (nonextractable) 3H-residues. The amounts of bound residues were higher in autoclaved than in nonautoclaved and in sandy than in either heavy or light soils. The mono- and didealkylated products were detected as well as unknown polar derivatives. 3H-residues bound to preextracted soils were absorbed by bean plants. The roots contained (0.20 - 0.72 ppm) more residues than did the shoots (0.07 - 0.15 ppm) and the uptake was higher in the case of autoclaved especially sandy soils.