The multiplicative model for cancer risk assessment: applicability to acrylamide

B. Paulsson, F. Granath2,, J. Grawé1,, L. Ehrenberg1, and M. Törnqvist,3

Department of Environmental Chemistry and
1 Department of Genetic and Cellular Toxicology, Stockholm University, S-106 91 Stockholm, Sweden and
2 Department of Medical Epidemiology, Karolinska Institute, S-171 77 Stockholm, Sweden


    Abstract
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 Abstract
 Introduction
 References
 
According to a multiplicative model for prediction of cancer risk for genotoxic agents the incremental cancer risk is, for low–intermediate exposures, proportional to target doses of the genotoxic substance and to the background risk in control groups. This model has been applied to evaluate cancer tests of acrylamide in rodents. Because of its reactivity toward DNA, glycidamide is assumed to be the causative genotoxic metabolite of acrylamide. Evaluation of experimental data according to the multiplicative model shows that mice, compared with rats, are of the order of 10 times more sensitive per administered dose of acrylamide. The US EPA procedure would, however, generally predict rats to be about twice as sensitive as mice to carcinogenic chemicals, because their estimates are based on scaling of the dose per square meter body surface area, as a surrogate for metabolic differences between the species. The comparison of rats and mice with respect to observed cancer incidence is at a key position in the evaluation of the usefulness of risk models for extrapolation between species. In the present study mice and rats were compared, with respect to in vivo doses of acrylamide and the metabolite glycidamide, after exposure to acrylamide. The relative in vivo doses were inferred from levels of hemoglobin adducts. The adduct levels from glycidamide were, per administered dose of acrylamide, ~3-10 times higher in mice than in rats. In combination with the above mentioned higher sensitivity of mice than rats in cancer tests of acrylamide this is compatible with the concept that glycidamide is the key genotoxic factor in acrylamide exposure. Furthermore, it is shown that the multiplicative, i.e. relative, risk model and measurements of the dose of the genotoxic factor give good prediction of the observed risk from acrylamide in cancer tests with rats and mice.

Abbreviations: AA, acrylamide; DD, doubling dose; GA, glycidamide; GC-MS/MS, gas chromatography–tandem mass spectrometry; GSH, glutathione; PFPTH, pentafluorophenylthiohydantoin.


    Introduction
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 Abstract
 Introduction
 References
 
Acrylamide (AA) is a reactive vinyl monomer used in the synthesis of polyacrylamide products, inter alia as a flocculent for waste water treatment, as a grouting agent, as a soil stabilizer, as an additive in paper and textiles, etc. AA can be absorbed into the body through the skin, by inhalation and via the gastrointestinal tract. AA is neurotoxic, with effects on both the central and peripheral nervous systems, is a skin and airway irritant, gives rise to increased cancer incidence and reproductive disruption in animal tests and is classified as probably carcinogenic in humans (1). The biological half-life of free AA in humans is estimated at ~4.6 h (2). AA is mainly metabolized by conjugation with glutathione (GSH) (3), but also by P450 enzymes to glycidamide (GA) (ref. 4; Figure 1Go), which is assumed to be the genotoxic factor in AA exposure. In contrast to AA, GA has been shown to form stable adducts with DNA in mice and rats after AA treatment and mice seem to have higher GA adduct levels than rats (5). Experimental data show that, with respect to tumor induction, mice are more sensitive to AA than rats (1,6).



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Fig. 1. Metabolism of acrylamide (AA) to glycidamide (GA) in vivo

 
A multiplicative model, linear at low–intermediate doses, has been proposed for prediction of cancer risk for genotoxic chemicals (7). This model is already in use for cancer risk projection of exposure to ionizing radiation. According to this model the incremental cancer morbidity or mortality, denoted {Delta}P, is proportional both to the dose (D) and to P0, i.e. the total or site-specific background cancer risk,


Dose means the accumulated lifetime dose of the genotoxic factor in the target organs. The risk coefficient ß is approximately the same for different sites and, as has been shown for radiation, presumably also for different species (7). For {gamma}-radiation and X-rays ß is ~0.4% of P0 per rad for mice, dogs and humans. In consequence, the total tumor incidence at responding sites gives a safer measure of the risk than data for individual cancer types and, furthermore, the model facilitates interspecies extrapolation if properly defined doses are used.

In contrast to a multiplicative model, most models used are additive, i.e. the risk increment, which is proportional to dose, is independent of and added to the background risk, P0.

For published cancer test data this model has been evaluated for several chemicals, including AA (7). For chemicals, however, a dose can be defined at different levels: exposure dose, absorbed dose, in vivo dose and target dose (8). Absorbed dose is given in mg or mmol per kg body wt and if multiplied by the in vivo lifespan of the chemical or of a formed reactive metabolite the in vivo dose is obtained. The in vivo dose is defined as the time integral of concentration (`area under the curve'), which is comparable with radiation doses expressed as energy absorbed per unit mass. The dose in the blood is often used as the in vivo dose, expressed as levels of adducts to blood proteins or to DNA in leukocytes. The dose in the target organs, the target dose, may in many cases be estimated from the dose in the blood.

For extrapolation between species the multiplicative model has to be based on the in vivo dose. However, for evaluation of the applicability of the model to published cancer tests the administered/absorbed dose, if proportional to the in vivo dose, could be used. The sensitivity may then be expressed as the inverse of the doubling dose (DD), e.g. the dose that leads to risk increments as great as P0.

The evaluation of published cancer test data on AA showed that the lifetime absorbed DD for mice was 20–50 mg/kg body wt and for rats ~500 mg/kg body wt (Table IGo). This means that, with respect to tumor induction, mice are 10–20 times more sensitive compared with rats per unit absorbed lifetime dose of AA (Table IIGo). As shown in the footnote to Table IIGo, it is possible that this sensitivity ratio of 10–20 should be reduced to 3–10, considering the effect of age at exposure.


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Table I. Sensitivities for cancer of mice and rats after acrylamide treatment, expressed as the lifetime absorbed doubling doses (DD, mg/kg body wt). DD values were calculated according to the multiplicative model, from published animal cancer studies 6
 

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Table II. Relative glycidamide (GA) doses in acrylamide (AA)-treated rats and mice, compared with relative sensitivity/cancer risk inferred from the calculated DD values in Table IGo
 
If the multiplicative model is valid for interspecies extrapolation the relative sensitivity should be proportional to the relative in vivo dose of the genotoxic factor. In the present study mice and rats were compared, with respect to in vivo doses of AA and the putatively genotoxic metabolite GA, after exposure to AA. The relative in vivo doses were obtained from levels of hemoglobin adducts.

Male mice (strain CBA; B&K Universal, Sollentuna, Sweden) and male rats (Sprague–Dawley; Charles River Sverige AB, Uppsala, Sweden), 7–8 weeks old, were used. The animals were treated with AA (acrylamide for electrophoresis; Merck, Darmstadt, Germany) by i.p. injection with doses of 0.3–1.5 mmol/kg body wt, using between two and four animals per dose. In each experiment three or four control animals were used. Blood for analysis was collected 24–48 h after injection. The blood samples were analysed according to the N-alkyl Edman method, a method for mass spectrometric determination of adducts to N-terminal valines of hemoglobin (9). After treatment with the Edman reagent, pentafluorophenyl isothiocyanate, the adducts are detached from hemoglobin as pentafluorophenylthiohydantoins (PFPTHs), N-(2-carbamoylethyl)-L-valine-PFPTH and N-(2-carbamoyl-2-hydroxyethyl)-L-valine-PFPTH from AA and GA, respectively, which were analysed by gas chromatography–tandem mass spectrometry (GC-MS/MS). The AA adduct levels were quantified and the levels of GA adducts were estimated relative to the AA adduct levels by comparing the respective peak areas in the GC-MS/MS chromatograms.

The results show differences in adduct levels in the two species. The AA adduct level per administered amount of AA was ~3 times higher in rats than in mice. The relative adduct levels of GA per administered amount of AA was 3–10 times higher in mice than in rats (Table IIGo). These differences are evidently consequences of the metabolism of AA to GA being more efficient in mice than in rats.

Related differences in AA metabolism between mice and rats have been shown in an in vivo study by Sumner et al. (3). After per os administration of AA (and acrylonitrile) rats had higher levels of GSH-conjugated AA in the urine compared with mice, while the levels of free and GSH-conjugated GA were higher in mice than in rats.

Reactivity of the N-terminal valine in hemoglobin is expected to be approximately the same for rat and mouse globins (7); therefore, the ratio of adduct levels in these proteins will reflect the ratio of in vivo doses. Thus the relative in vivo dose of GA, per administered dose of AA, was found to be ~3–10 times higher in mice than in rats. Strictly speaking this factor refers to doses in blood. Considering the rapid distribution (10) and relatively long lifespans (2) of AA and GA in the body the same factor is with some certainty approximately valid for target doses as well.

Table IIGo shows the relative GA doses and the relative sensitivity to cancer (cancer risk) evaluated according to the multiplicative model for mice and rats after exposure to AA. Comparison of the data shows agreement between the relative in vivo GA dose levels per administered dose of AA and the experimentally found relative cancer risk.

The present results are in agreement with GA being the genotoxic agent in exposure to AA. The finding that the inter-species ratio of sensitivities is compatible with the ratio of GA doses gives, furthermore, support to the coefficient ß in the risk model (see above) being approximately the same in different species, as indicated for ionizing radiation (7). It also supports the applicability of the multiplicative model for cancer risk assessment. This model is based on the lifetime dose of the (predominant) genotoxic compound or metabolite and the relative increment (above the background, P0) in the total incidence of tumors at responsive sites.

In a published quantitative cancer risk assessment of AA by the US EPA (11) extrapolation from rats to humans was performed using a linear model for risk assessment, with a trans-species conversion based on scaling of the dose per square meter body surface area, as a default surrogate for metabolic differences. If the body surface area is assumed to be proportional to the body mass raised to the power of 2/3, the conversion factor between two species with average body weights W1 and W2 will be equal to (W1/W2)1/3. Using this procedure, male rats (average weight 300 g) would be approximately twice as sensitive as male mice (average weight 25 g). This points in the opposite direction, as compared with observed cancer incidence ratios in tests of AA, as well as certain other chemicals, e.g. butadiene (12).

Measurement of in vivo dose of the reactive intermediate in the test animal, as well as in humans, will improve the reliability of the calculated risk coefficient and would be in line with the revised recommendations of the US EPA (13) to use mechanism-based models in cancer risk assessment. The suggested model aims at a simple and manageable description of the multiplicative interaction of induced increase in mutation frequency with the existing pattern of inherited or acquired growth-promoting conditions. The dose (as defined above) in target tissues is the prime determinant of incremental mutation frequency. Doses, as calculated from measured levels of specific stable adducts to a macromolecule with known turnover kinetics, implicate the net effects of all enzymatic, chemical and physiological factors on rates of formation and disappearance of reactive chemicals or metabolites. For risk estimation of AA in humans in vivo doses of GA are evidently essential, the risk being estimated either by comparison with rodent incidence per unit dose GA or via the radiation dose equivalence of GA doses (7). The absolute value of in vivo GA dose at a given AA exposure is still uncertain.


    Notes
 
3 To whom correspondence should be addressed Email: margareta.tornqvist{at}mk.su.se Back


    Acknowledgments
 
Thanks are due to Dr Emma Bergmark for providing reference compounds and to Mr Ioannis Athanasiadis for skilful technical assistance in mass spectrometric analysis. We acknowledge economic support from The Swedish Council for Work Life Research and from The Swedish National Board for Laboratory Animals.


    References
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 Abstract
 Introduction
 References
 

  1. IARC (1994) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, 60, Acrylamide. IARC, Lyon, pp. 389–433.
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  14. The National Research Council's Committee on the Biological Effects of Ionizing Radiations (BEIR) (1990) Health Effects of Exposure to Low Levels of Ionizing Radiation, BEIR V. National Academic Press, Washington, DC, pp. 161–241.
Received July 27, 2000; revised January 12, 2001; accepted January 15, 2001.





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