Potency grading in carcinogen classification.

In 1992 the United Nations Conference on Environment and Development decided to harmonize carcinogen classification systems. A proposal for a harmonized classification system is currently being considered by the Organization for Economic Cooperation and Development (OECD). In many countries, classification of a chemical as carcinogenic triggers labeling requirements. Implicit in the labeling requirements are often restrictions on the sale of consumer products and workplace regulations. Many of the current classification systems for carcinogens use a single concentration limit for the minimum concentration of a carcinogen in a preparation (mixture) that requires labeling. For high-potency carcinogens, one concentration limit may not adequately express the hazard, whereas for low-potency carcinogens, one limit may overestimate the hazard caused by the carcinogen in the preparation (mixture). The potency grading system discussed consists of three potency groups: high-, medium-, and low-potency carcinogens. It is envisioned that the different classes will trigger different labeling requirements. In the process of potency grading, a preliminary conclusion as to whether a substance shows high, medium, or low potency is initially based on a tumorigenic dose descriptor. The preliminary potency evaluation may then be modified after due consideration of a number of additional elements. These may include evaluation of the dose-response curve; site-, species-, strain-, and sex-specific activity; mechanisms including genotoxicity; mechanistic relevance to humans; toxicokinetics; and other factors. The potency grading system discussed is applicable to most carcinogen classification systems, including that currently being considered by the OECD.

T25: a simplified carcinogenic potency index: description of the system and study of correlations between carcinogenic potency and species/site specificity and mutagenicity.

A simplified carcinogenic potency index, the T25, is proposed as a practical method for the inclusion of potency considerations in carcinogen classification systems. The T25 is the chronic daily dose in mg per kg bodyweight which will give 25% of the animals tumours at a specific tissue site, after correction for spontaneous incidence, within the standard life span of that species. Calculated T25 values of a set of 113 US National Cancer Institute/National Toxicology Program (NC/NTP) carcinogens showed excellent correlation (correlation coefficient 0.96, P < 0.0001) with the carcinogenic potency index TD50 of Peto et al. (1984). The mean of T25 values for 51 transspecies, multiple common site NCI/NTP carcinogens were 10-fold lower than those for 62 NCI/NTP single species, single site carcinogens. For these 113 carcinogens, the mean T25 values were approximately 3-fold lower for agents that were also mutagenic in Salmonella compared to the non-mutagenic agents.

Carcinogen classification systems: similarities and differences.

An overview of regulatory classification systems on carcinogens in the Organization for Economic Cooperation and Development (OECD) countries is presented based on a questionnaire study. Most OECD countries have implemented legislation including classification systems and lists of carcinogens. Basically, there are two types of classifications systems. The major difference between the two is that in one system carcinogens are classified according to the weight of evidence for carcinogenic effects in humans, whereas in the other carcinogens are allocated to various groups according to potency. Even if the classification systems may differ, the substances classified as carcinogens are to a large extent the same. Classification of carcinogens will in many countries require hazard labeling. This labeling, i.e., the limit for labeling of substances and preparations, and risk phrases show considerable similarities, but differ in certain aspects. Several countries have restrictions on sale and/or use of carcinogens. There is a trend toward introducing more mechanistic considerations in the classification of carcinogens.

Uniform system for the evaluation of substances. I. Principles and structure.

In April 1994, the first version of the Uniform System for the Evaluation of Substances (USES 1.0) was launched to comply with an action point of the Netherlands National Environmental Policy Plan. USES is a tool for the rapid, quantitative assessment of the hazards and risks of chemical substances, including new substances, existing substances, agricultural pesticides and biocides. It was developed to be applied as a decision-support system by the central government, by industry and institutes in the private sector and by international fora. Since hazard and risk assessment must be transparent to all users and easy to perform, USES 1.0 is well documented and available as a computer program. An overview of this system will be presented including its objectives, the national and international framework, the general principles involved, as well as the structure and the content of the models used.

Classification of chemicals for carcinogenic and mutagenic properties.

In the framework of the EC labelling guide, three categories were devised for carcinogenic as well as for mutagenic chemicals. In short, category 1 is for compounds shown to produce these effects in man, whereas category 2 is meant to contain substances that should be regarded as such, generally on the basis of sufficient experimental data. Category 3 is for compounds that cause concern but for which the evidence is not sufficient for 1 or 2. These general statements have been implemented, both for carcinogenicity and mutagenicity, by a set of criteria and considerations of the group of "specialized experts" who are supporting the "national experts" in deciding upon the classification of individual compounds. Essentially, this classification relies on the strength of the experimental or epidemiological evidence rather than on considerations of risk. In other words, it is based on proven intrinsic properties of the substances. Compared to the IARC classification for carcinogens, the EC criteria rely more on mechanistic data and on qualitative considerations concerning the relevance for man, and less on a strict count of the amount of positive experimental evidence. Difficulties of this (and any) classification system include: (1) the problem of classifying with limited data and (2) the fundamental problem involved in forcing sets of multi-facetted, and often unsatisfactory, scientific data into a simple one-dimensional scheme that may have huge economic consequences.

Short-term occupational exposure limits: a simplified approach.

This paper presents a simplified proposal for setting health standards based on short-term exposure limits (STEL). It presents an alternative to the approach by the German MAC Commission: with only three instead of five categories, no fixed excursion factors, but ranges; more restrictive duration of sampling; no fixed frequencies of the number of accepted excursions per workshift.

Glycoprotein metabolism in skin cancer: synthesis, pool size, and partial characterization of glycoproteins in the rat basosquamous cell carcinoma.

Uptake of fucose, glucosamine, galactose, and mannose and the incorporation of these sugars into glycoconjugates have been quantified in a model epithelial tumor, the basosquamous cell carcinoma of the rat. Following isolation of glycoprotein and papain digestion, the fucosylated glycopeptides were fractionated according to molecular weight (Mr) and by affinity chromatography. Analysis of cellular material revealed a 2- to 3-fold reduction in the proportion of high-Mr fucopeptides synthesized by the tumor compared with normal epidermis, accompanied by a profound block in the incorporation of galactose into glycoconjugates. Parallel investigation of carbohydrate removed from the cell surface with trypsin or spontaneously shed into the medium indicated a striking decrease in the total release of fucopeptides from the tumor; the Mr was (respectively) normal or increased. Thus the fucopeptide abnormality appears to be accounted for almost entirely by a shift toward accumulation of low-Mr material at internal locations within the cell.

Glycoprotein composition of psoriatic epidermis in relation to growth control.

The composition of isolated [3H]-fucose- and [3H]-glucosamine-labeled glycopeptides from psoriatic lesion, psoriatic uninvolved, regenerating-normal, and normal epidermis (subdivided into basal and differentiated cells) has been analyzed according to molecular weight and affinity to concanavalin A. Basal cells from normal skin have a higher sialic acid content of their "biantennary" fucose-labeled glycoproteins than differentiated cells. Fucose-labeled glycoproteins from regenerating normal skin show an increased molecular weight of their carbohydrate moieties which precedes entrance of the cell into S-phase. In psoriatic uninvolved skin, glycoproteins with more highly branched carbohydrate structures are present in reduced quantity. The previously reported increase in percentage of high-molecular-weight fucose-labeled glycopeptides for psoriatic lesions appears to be specific in that its composition cannot be explained in terms of an increased growth fraction.

Studies on the plasma membrane of normal and psoriatic keratinocytes. 6. Cell surface and shed glycoproteins.

Quantification and characterization of [3H]-fucose-labeled cell surface glycoproteins are reported. Two approaches have been compared; first the analysis of glycoprotein shed spontaneously into the medium during incubation of keratinocytes in vitro, and second the study of material released by exposure of the cell to proteolytic enzymes. It is shown that psoriatic keratinocytes "shed" glycoprotein more rapidly than normal, although the material is of similar molecular weight (mainly "biantennary" transferrin type glycopeptides). By contrast, the percentage of glycoprotein released by proteolysis of psoriatic keratinocytes is normal, but the molecular weight distribution of the labeled glycopeptides is markedly altered. The abnormal turnover and composition of fucose-labeled glycoproteins from the cell surface may be related to the loss of growth control in psoriatic epidermis.

Cutaneous sialidase.

A fluorometric microassay is described for sialidase using the natural substrate sialyllactose. This technique has been used to characterize and quantify cutaneous sialidase. The enzyme was found exclusively in the particulate fraction of skin homogenates. Its properties were similar to those from other mammalian tissues, showing a pH optimum of 4.0 and a Km of 0.47 mM. Although the modal value of levels in psoriatic biopsies was similar to normal, a few specimens contained exceptionally high activity.

Studies on the plasma membrane of normal and psoriatic keratinocytes. 5. Lectin binding.

The glycocalyx of epidermal keratinocytes from psoriatic patients has been investigated by means of lectins. Striking changes were found in the levels of glucose and/or mannose (concanavalin A) and of N-acetylglucosamine and/or sialic acid (wheat germ agglutinin) on the surface of cells from the psoriatic lesion. Smaller but significant changes were seen in the clinically uninvolved epidermis of the patient. A marked increase in the affinity of the cell surface for Ulex europus agglutinin (fucose-specific) confirms our previous reports of structural alterations in fucose-containing oligosaccharides in psoriasis.

Studies on the plasma membrane of normal and psoriatic keratinocytes. 4. Characterization of glycoconjugates.

A substantial proportion (20-50%) of radioactive sugar incorporated into glycoconjugates by normal human keratinocytes was soluble in chloroform-methanol; using (14C)-galactose as precursor about half of this fraction was neutral lipid. The incorporation of labelled sugars into the lipid fraction was consistently increased in keratinocytes derived from psoriatic lesions. The abnormality in fucose metabolism which we reported previously has been confirmed. In particular we have shown that the molecular weight of fucosylated glycopeptides appears to be abnormally high in the untreated psoriatic lesion, possibly reflecting an increased degree of branching. In psoriatic uninvolved epidermis and in treated psoriatic lesions the situation is reversed, the molecular weight being significantly lower than normal.

Studies on the plasma membrane of normal and psoriatic keratinocytes. 3. Uptake of labelled sugars and their incorporation into glycoconjugates.

We report the uptake of four labelled sugars by keratinocytes isolated from normal epidermis, psoriatic 'uninvolved' skin and psoriatic lesions. Our findings include the following: (1) The rate of uptake of all sugars by the psoriatic lesion is increased. (2) This abnormally high uptake diminishes dramatically during 22 h incubation in vitro. (3) There is a striking abnormality in the metabolism of fucose by psoriatic keratinocytes; our data suggest an increased rate of incorporation of fucose into glycoconjugates.

Studies on the plasma membrane of normal and psoriatic keratinocytes. 2. Cyclic AMP and its response to hormonal stimulation.

Cyclic AMP levels have been determined for the first time in isolated keratinocytes. Values were more reproducible than those reported using epidermal slices. Evidence is presented to show that damage to hormone receptors is minimal. Other observations include the following: (1) Keratinocytes from psoriatic lesions showed reduced 'resting' levels of cyclic AMP as well as a diminished response to adrenaline. (2) Cyclic AMP levels were maximal in the basal cells, falling dramatically in fully differentiated keratinocytes. (3) The topical application of a corticosteroid (fluocinolone acetonide) did not modulate the response of adenyl cyclase to hormonal stimulation.

Studies on the plasma membrane of normal and psoriatic keratinocytes. I. Preparation of material and morphological characterization.

A method is described for the preparation of isolated keratinocytes suitable for subsequent biochemical studies. Scanning electron microscopy showed that the maturation process is accompanied by an increase in cell size and a shortening and eventual loss of microvilli. Psoriatic keratinocytes are distinguishable by exhibiting longer microvilli at all levels of maturation.