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.

Optimal parameters for laser tissue soldering: II. Premixed versus separate dye-solder techniques.

BACKGROUND AND OBJECTIVE: Laser tissue soldering by using an indocyanine green (ICG)-doped protein solder applied topically to the tissue surface and denatured with a diode laser was investigated in Part I of this study. The depth of light absorption was predominantly determined by the concentration of the ICG dye added to the solder. This study builds on that work with an in vitro investigation of the effects of limiting the zone of heat generation to the solder-tissue interface to determine whether more stable solder-tissue fusion can be achieved. STUDY DESIGN/MATERIALS AND METHODS: An alternative laser tissue soldering technique was investigated, which increased light absorption at the vital solder-tissue interface. A thin layer of ICG dye was smeared over the surface to be treated, the protein solder was then placed directly on top of the dye, and the solder was denatured with an 808-nm diode laser. Because laser light at approximately 800 nm is absorbed primarily by the ICG dye, this thin layer of ICG solution restricted the heat source to the space between the solder and the tissue surfaces. A tensile strength analysis was conducted to compare the separate dye-solder technique with conventional techniques of laser tissue soldering for which a premixed dye-solder is applied directly to the tissue surface. The effect of hydration on bond stability of repairs formed by using both techniques was also investigated using tensile strength and scanning electron microscopy analysis. RESULTS: Equivalent results in terms of tensile strength were obtained for the premixed dye-solder technique using protein solders containing 0.25 mg/ml ICG (liquid solder, 220 +/- 35 N/cm(2); solid solder, 602 +/- 32 N/cm(2)) and for the separate dye-solder technique (liquid solder, 228 +/- 41 N/cm(2); solid solder, 578 +/- 29 N/cm(2)). The tensile strength of native bovine thoracic aorta was 596 +/- 31 N/cm(2). Repairs created by using the separate dye-solder technique were more stable during hydration than their premixed dye-solder counterparts. The conventional premixed dye-solder was simpler and approximately twice as fast to apply. The separate dye-solder technique, however, increased the shelf-life of the solder, because the dye was mixed at the time of the experiment, thus conserving its spectral absorbency properties. CONCLUSION: Two laser-assisted tissue soldering techniques have been evaluated for repairing aorta incisions in vitro. The advantages and disadvantages of each of these techniques are discussed.

New myrsinol-related polyfunctional pentacyclic diterpene esters from roots of Euphorbia prolifera.

Five Euphorbia substances, SPr1-SPr5, were isolated from the roots of Euphorbia prolifera. They were found to have similar structures but were inactive in a mouse ear inflammation assay. By nmr analysis and after single-crystal X-ray crystallography the structure of SPr5 was established as a hexaester (tetraacetate-benzoate-propionate) of a hitherto unknown polyfunctional pentacyclic diterpene parent alcohol, structurally related to myrsinol. As judged from its nmr spectra, SPr4 is an analogue of SPr5, carrying an isobutyrate substituent in place of a benzoate ester functionality. SPr1-SPr3 were partially characterized by their mass spectra as esters of diterpene parent alcohols possibly related to the myrsinol structure. SPr1-SPr5 may represent one of the product lines branching off the proposed main route of biogenesis of the oligocyclic diterpenoid skin irritants and tumor promoters occurring in many, but not all, of the species in the plant families Thymelaeaceae and Euphorbiaceae.

Improved access to highly unsaturated skin irritants of the daphnane type from latex of Excoecaria oppositifolia.

A new and more efficient procedure was developed to obtain 5 beta-hydroxyresiniferonol-6 alpha,7 alpha-epoxide- 9,13,14-ortho(2,4,6-decatrienoate) [Excoecaria factor O1] by alkaline transesterification of corresponding 20-ester(s) genuinely present in latex of Excoecaria oppositifolia. It was obtained in a 10-fold better yield than previously, probably due to speeding up the separation procedures of the sensitive compound. O1 is an appropriate starting material for the preparation of the daphnane prototype irritant and tumor-promoting orthoester simplexin (SIM) especially of a multiple tritium-labeled form thereof. Moreover, a new Excoecaria factor O3, i.e. the 9,13,14-ortho(2,4,6,8-hexadecate-traenoate) of 5 beta,12 beta-dihydroxyresiniferonol-6 alpha,7 alpha-epoxide was isolated after alkaline transesterification of a TLC-uniform fraction, composed of corresponding 20-ester(s).

A skin irritant principle from Euphorbia matabelensis Pax.

A diterpene of the ingenane-type parent alcohol with tetradecanoic acid as the acid substituent was isolated by chromatographic methods from the latex of Euphorbia matabelensis. The ingenol ester exhibited irritant activity on the mouse ear.

A skin irritant phorbol ester from Euphorbia cooperi N E Br.

From the fresh latex of Euphorbia cooperi N E Br was isolated by partition and chromatographic methods, a diterpene ester 12-deoxyphorbol-16-isobutyrate-13-tigliate. The phorbol ester exhibited highly irritant activity on the mouse ear. Since skin irritancy is an indication of possible tumour promotion, the use of this plant as a medicine should be discouraged.

Structure Activity Relations of Polyfunctional Diterpenes of the Tigliane Type, VI1.

A method for the isolation and preparation of 12-deoxyphorbol ( 1) from Euphorbium resin ( EUPHORBIA RESINIFERA Berg) was established to provide substantial amounts of 1-esters for bioassays. Acylation of 1 yielded 12-deoxyphorbol-13,20-diesters ( 2, esters with acids CH (3)(CH (2)) (n)COOH, n = 0, 4, 8, 12, 16, 20; and with benzoic, oleic, elaidic and linoleic acid). Upon mild transesterification the 13,20-diesters 2 yielded the 13-monoesters 3. Both ester types were tested for their irritant activities, the 13-monoesters were also assayed for their tumor-promoting activity. A dependance of both of these activities on the chain length of the acyl residues is noticeable. For both probably a broad maximum exists around the tetradecanoate.

Skin tumor promotion by phorbol esters is a two-stage process.

In the semisynthetic compound phorbol 12-retinoate 13-acetate (PRA), the antipromoting principle of vitamin A acid is combined with the structure of a phorbol ester tumor promoter. In skin of NMRI mice, a single topical application of PRA induces skin inflammation, epidermal proliferation, and sustained hyperplasia to a similar extent and apparently along the same pathway as an equimolar dose of the strong tumor promoter phorbol 12-myristate 13-acetate (PMA). The mitogenic effects of both PRA and PMA are mediated by prostaglandin E synthesis. However, in mouse skin initiated with 7,12-dimethylbenz[a]anthracene, PRA does not promote tumor development, even at a high dose. Under continuous PRA treatment, however, one to four applications of PMA (insufficient by itself to promote tumor growth) gave a strong tumor response. Thus, it can be demonstrated that the effects necessary for tumor promotion can be brought about by a single application of PMA and that the subsequent chronic hyperproliferation of epidermis is probably necessary only to make the tumors visible. By using the nonpromoting irritant mitogen PRA, the concept of two-stage tumor promotion can thus be strongly supported. Furthermore, in the NMRI mouse, PRA is a much more potent second-stage promoter than mezerein, recently reported to be an incomplete promoter in the Sencar mouse.