* Department of Biology/Toxicology, 401 College Avenue, Ashland University, Ashland, Ohio 44805; and
Institute of Pharmacology and Toxicology, University of Graz, Graz, Austria
Received May 30, 2000; accepted July 31, 2000
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ABSTRACT |
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Key Words: chemical mixture toxicity; dose-addition; independence; copper sulfate; FETAX.
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INTRODUCTION |
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The complexity of the cross-linking process leads to several possible mechanisms for induction of osteolathyrism. First is inhibition of LO by direct, irreversible binding of the agent to the enzyme, leading to a decrease in enzyme activity (Tang et al., 1983). Second, binding of the chemical to aldehyde precursors of developing fibers could inhibit further cross-linking reactions (Deshmukh and Nimni, 1969
). A third possible mechanism is that the osteolathyrogen may interfere with cofactor generation and/or binding (Bird and Levene, 1983
; Kagan and Trackman, 1991
). Another potential mechanism involves the chelation of copper (Harris et al., 1974
; Osterberg, 1980
). It has also been proposed that osteolathyrism could be due to oxidative stress (Ghate, 1985
).
When exposed to sufficient concentrations of osteolathyrogens, frog embryos show abnormal notochord development. At the lowest effective concentrations, osteolathyritic lesions are small dorsoventral bands and/or an outpocketing of the notochord at its ventral margin. At higher concentrations, there is greater disorganization within the notochord, producing a "wavy-tail" appearance (Riggin and Schultz, 1986; Schultz et al., 1985
, 1988
). Light and electron microscopy have further characterized the effects as disorganization of collagen fibers and, for some chemicals, absence of the elastic externa (Riggin and Schultz, 1986
; Schultz et al., 1985
).
ß-aminopropionitrile (ßAPN) is an osteolathyrogen (Levene, 1961) that reportedly induced cross-linking defects by binding to the active site of LO, as the amount of ßAPN bound was proportional to the amount of enzyme inactivation (Tang et al., 1983
). Frog embryos exposed to this nitrile in several labs (Bantle et al., 1996
; Dawson and Wilke, 1991
) showed abnormal notochord morphology similar to that observed for ureides, acid and benzoic acid hydrazides, and alkyl carbazates (Dawson et al., 1990
; 1991
; Schultz and Ranney, 1988
; Schultz et al., 1985
, 1988
).
In other studies involving frog embryos, certain dithiocarbamates were found to be potent developmental toxicants (Bancroft and Prahlad, 1973). At lower concentrations, they disrupted normal notochord development. At higher concentrations, the notochord was severely malformed and other abnormalities could be induced (Ghate and Mulherkar, 1980
). Diethyldithiocarbamate (DTC) is a copper chelator (Harris et al., 1974
; Osterberg, 1980
). Because of its strong affinity for copper, DTC may alter LO activity, thereby adversely affecting connective tissue cross-linking. However, when the interaction between DTC and cysteine was analyzed, it was concluded that the developmental effects caused by DTC were due to oxidative stress (Ghate, 1985
).
The frog embryo mixture toxicity assay (Dawson and Pöch, 1997) determines the toxicity of binary chemical mixtures through statistical comparison of the mixture-response curves with two mechanistic combined effects models. As a result, the approach may be useful in relating the combined toxicity to common or different mechanisms of chemical action. To initially evaluate the mixture toxicity testing strategy and data analysis techniques developed, and to determine the value of the approach for distinguishing between chemicals that work by the same or by different mechanisms, a series of osteolathyrogen combinations is being tested. Osteolathyrism was selected as the toxic effect for evaluation because of the possibility that osteolathyrism results through more than one mechanism (i.e., enzyme inhibition, copper chelation, cofactor disruption, etc.); because frog embryos exposed to osteolathyrogens exhibit characteristic lesions in the notochord that are easily detected in the transparent tadpoles using a dissecting microscope; and because exposed frog embryos exhibit osteolathyritic lesions at chemical concentrations that do not typically induce other malformations or death, thereby reducing the chance that confounding toxicity factors affect data interpretation. In this study, the osteolathyrogens ßAPN and DTC were tested to determine their combined osteolathyritic effects. In addition, DTC was tested with copper sulfate to determine the importance of copper in DTC-induced osteolathyrism. Results of similar tests of ßAPN with copper have been reported (Dawson et al., 2000
).
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MATERIALS AND METHODS |
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Assay procedures.
FETAX solution was used for breeding and as the diluent for all other embryo exposures. Following breeding, embryos were collected and the jelly coat was removed by gentle agitation in a 2% (w/v) cysteine solution (pH 8.1) for 12 min. After rinsing in FETAX solution (Dawson and Bantle, 1987), dead and abnormal embryos were discarded, with remaining embryos microscopically examined for proper development in blastula and early gastrula stages. Those judged to be developing properly were retained for testing.
ß-aminopropionitrile (ßAPN, CAS # 2079-89-2, a monofumarate salt) and diethyldithiocarbamate (DTC, CAS # 20624-25-3, sodium salt trihydrate) were purchased from Sigma Chemical Co. (St. Louis, MO). All reported concentrations of ßAPN and DTC were corrected for fumarate or for sodium and water content, respectively.
Individual stock solutions for ßAPN and DTC were prepared, and the pH was adjusted to 7.3 as needed. Thirty-six treatment solutions at 25 ml were prepared based on a 1.2-factor matrix design (Dawson and Pöch, 1997), resulting in 1 control, 12 single-chemical (6 per chemical), and 23 mixture treatments (Fig. 1
). This design produces 2 single-chemical and 7 mixture-response curves: 2 with the ßAPN concentration fixed and the DTC concentrations increasing, 2 vice versa, and 3 fixed-ratio curves (1:1, 1:3, 3:1) in which the concentrations of both chemicals increase.
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For the DTC:copper test, a 500 ml stock solution of cupric sulfate-pentahydrade (CAS # 7758-99-8, Fisher Scientific) was prepared in FETAX solution. There were 32 treatments consisting of 1 control, 7 DTC-only, 3 copper sulfate-only (0.5, 1.0, and 1.5 mg/l), and each of those 3 copper sulfate concentrations with each of the 7 DTC concentrations (i.e., 21 mixture treatments). For this combination there were 60 embryos per treatment divided equally between three dishes; with each dish containing 8 ml of solution. The copper sulfate solution was used for treatment preparation throughout the test. Other than the changes noted here, procedures were as described for the ßAPN:DTC test.
Spectrophotometry.
Because DTC is a metal chelator and there was color formation when DTC and copper sulfate were combined, spectrophotometry was employed to examine aspects of the color formation. Spectrophotometric readings of DTC, ßAPN, and copper sulfate solutions, alone and in combination, were conducted using a Milton Roy Spectronic 1201 UV-visible scanning spectrophotometer.
Data analysis.
In order to determine if non-osteolathyritic malformations were related to chemical exposure, two multiple comparison tests were conducted. Dunnett's test (Dunnett, 1955) and the Student-Newman-Keuls test (Steel and Torrie, 1980) were used to analyze for differences (p
0.05) in rates of non-osteolathyritic malformations between each treatment and the control and across all treatments, respectively.
Experimental data points for osteolathyrism induced by chemical A alone and by chemical B alone were fit to sigmoidal curves using a four-parameter logistic function and SigmaPlot (Jandel Scientific, San Rafael, CA). Curve fit parameters were: a, minimum effect, b, slope, c, EC50 osteolathyrism, and d, maximum effect. Then, for each experimental response curve, theoretical curves for the number of osteolathyritic embryos were calculated for the dose-addition and independence models in the following manners.
Theoretical dose-addition curve calculation for comparison with fixed-dose responses (e.g., Fig. 2) were based on the principle described by Pöch et al. (1990). This procedure was conducted using SigmaPlot (Jandel Scientific, San Rafael, CA) as detailed by Holzmann et al. (1999). In essence the theoretical curve is obtained by calculating dose x of chemical A with which the concurrently applied fixed-dose of chemical B was equieffective, using the equation x = {([(a - d)/(a2 - d)] - 1)*(cb)}1/b where a, b, c, and d are the parameters (defined above) for the curve of chemical A alone and a2 is the minimum effect for the curve of chemical A with the concurrently applied fixed-dose of chemical B. From the definition of dose-addition, the effects of chemical A alone are to be expected at the concentrations of A-x. Therefore, the response values for the theoretical dose-addition curves of chemical A correspond to simulated concentrations of A-x.
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Fixed-ratio response calculations required that chemical B concentrations be converted to equivalent concentrations of chemical A for each fixed ratio (i.e., 1:3, 1:1, 3:1) using the equation B*factor A/B (Pöch et al., 1997).
The 2 goodness-of-fit test was used to compare the experimental responses with the theoretical responses (Pöch, 1993
), following performance of an F-test to compare slope values generated for each chemical tested alone. The goodness-of-fit analyses were used to determine whether the combined effects differed significantly (p < 0.05) from the theoretical effects for dose-addition and for independence.
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RESULTS |
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For all treatments, the frequency of death was 1.6%, whereas the frequency of non-osteolathyritic malformations in surviving tadpoles was 2.4%. Non-osteolathyritic defects were of the eye (1.8%), gut (1.6%), or edema (1.3%). Neither the numbers of dead tadpoles nor tadpoles with non-osteolathyritic malformations were significantly different between each treatment and the control or across all treatments.
The concentration-response data for ßAPN alone and for DTC alone (Table 1) were used in calculating expected combined effects for the seven ßAPN:DTC mixture curves. An F-test indicated the slopes for ßAPN alone and DTC alone were significantly different (F = 41.75, p = 0, d.f. = 11). For ßAPN alone, the response curve slope was 5.27. The slope for the DTC-alone response curve was 2.08, but the curve appeared to be biphasic. An analysis of this curve showed the lower phase having a slope of 2.62 [with a minimum effect (Emin) of 0 and a maximum effect (Emax) of 71] and the upper phase a slope of 3.71 (with Emin = 37 and Emax = 98).
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As with the ßAPN:DTC test, the DTC-alone response curve in the copper test (slope = 2.01) appeared to be biphasic, with the lower phase having a slope of 2.88 (Emin = 1, Emax = 70) and the upper phase a slope of 3.43 (Emin = 41, Emax = 99).
The EC50 for osteolathyrism induced by DTC alone was 0.80 mg/l, whereas the EC50 values for DTC with 0.5, 1.0, and 1.5 mg/l copper sulfate increased to 1.23, 1.73, and 2.07 mg/l, respectively. Although toxicity was reduced by coadministration of copper at the lower DTC concentrations, this reduction in toxicity was less prominent at the higher DTC concentrations, as evidenced by the lack of a right shift at the top of the DTC-copper response curves (Table 5, Fig. 4
). Previous tests (Dawson, et al., 2000
) showed an inhibition of ßAPN-induced osteolathyrism upon copper coadministration. As with DTC, the ßAPN EC50 was increased, but in this case there was a right shift of the entire response curve as opposed to that observed with DTC and copper herein.
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DISCUSSION |
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The frog embryo mixture toxicity assay, a modification of FETAX (ASTM, 1991; Dumont et al., 1983
), was developed to relate the combined effects of chemicals to common or different mechanisms of toxicity (Dawson and Pöch, 1997
; Mentzer et al., 1999
). The assay incorporates the fixed-dose (Pöch, 1993
) and fixed-ratio (Dawson, 1994
) mixture toxicity testing strategies into a 1.2-factor treatment design. In this design, each chemical is tested alone at six concentrations, treatments 27 for chemical A (in this case ßAPN) and 813 for chemical B (i.e., DTC). These treatments were selected by assigning the average EC50 for osteolathyrism induced by each chemical alone (determined from previous testing) the value 1.0 (i.e., treatments 5 and 11 for chemicals A and B, respectively). The other treatments for each chemical alone were then determined by multiplying or dividing the EC50 by 1.2x. The 1.2-factor values for generating the concentrations (as given in Fig. 1
) are rounded (e.g., treatment #2 is 1/1.25 = 0.40). For a chemical with a slope between 2 and 8 (Dawson and Pöch, 1997
), as is obtained for osteolathyrogens tested on Xenopus, such concentrations cover, at minimum, the linear portion of the dose-response curve. The single-chemical dose-response curves provide the data needed to calculate theoretical mixture-response curves for both the dose-addition and independence models. The 23 mixture treatments, also generated using the 1.2-factor matrix, give seven experimental mixture-response curves (Fig. 1
), which are compared to the theoretical curves using the
2 goodness-of-fit test. The results of the comparisons, then, indicate whether the chemicals are likely to induce toxicity by the same or by different mechanisms.
In this study, all seven mixture-response curves were consistent statistically with theoretical effects calculated using the dose-addition model. This is the expected result for two chemicals that induce effects by the same mechanism. However, a more thorough analysis of the response curves and osteolathyrogen:copper data suggests a more complex combined effect:
To evaluate the apparent biphasic nature of the DTC-alone response curves, preserved tadpoles were reevaluated by lesion location along the length of the notochord. For ßAPN and DTC, initial lesions appear in different locations, with the ßAPN lesions being in the tail posterior to the cloacal outlet (i.e., approximately the posterior two-thirds of the notochord), and the DTC lesions being at or anterior to the cloacal outlet (i.e., roughly the anterior third of the notochord). However, with increasing ßAPN concentration, anterior lesions become apparent, whereas with increasing DTC concentration, posterior lesions become apparent. Addition of a second lesion type, for either chemical, has the effect of altering the slope of the response curve, as established by the initial lesions, thereby giving the curve a biphasic appearance. The important question resulting from this observation is whether location of the lesions represents a difference in the way (i.e., mechanism) notochordal connective tissue cross-linking is disrupted.
There is support in the recent literature for the idea that a single osteolathyrogen could disrupt connective tissue cross-linking in more than one way (Dawson et al., 2000). For example, copper is known to be needed for proper LO activity in connective tissue fiber cross-linking. Recent work indicates that it is likely that copper is also needed in the regeneration of the carbonyl cofactor associated with LO (Dooley, 1999
; Matsuzaki et al., 1994
; Smith-Mungo and Kagan, 1998
; Wang et al., 1996
), setting up the possibility that copper-sensitive osteolathyrogens could adversely affect connective tissue fiber cross-linking at two different points in the cross-linking process. The exact mechanism by which LO brings about cross-linking of developing collagen and elastin fibers has yet to be discerned (Smith-Mungo and Kagan, 1998
), so proposed noncopper-dependent osteolathyrogenic mechanisms are also possible.
It should be feasible to assess the hypothesis that differences in location of osteolathyritic lesions represent different mechanisms by which the chemicals act, simply by analyzing the mixture data broken out by lesion location to develop goodness-of-fit comparisons with the dose-addition and independence models of combined effect. For example, for two chemicals A and B, one could examine the goodness-of-fit of the A:B mixture-response curves using posterior lesions only, anterior lesions only, and posterior lesions for A, with anterior lesions for B (and vice versa), to assess similarity or differences in mechanisms. This was not attempted for the ßAPN:DTC data presented herein, because complete concentration-response curves for both lesion locations were not obtained for each chemical alone, and because the n of 50 embryos per treatment was judged to be insufficient for providing a rigorous assessment of the hypothesis. The idea will be evaluated in future tests of osteolathyrogen combinations using n of 80100 embryos per treatment to provide the desired statistical power. In addition, since several osteolathyrogens show notochord lesions in both locations, it will be necessary to examine this phenomenon across multiple combinations of osteolathyrogens, in osteolathyrogen:copper combinations to determine if one lesion location is inhibited more frequently than another when copper is present, and if coadministration of copper is shown to preferentially inhibit a specific lesion location, in tests in which binary osteolathyrogen combinations are examined in the presence and absence of copper.
A clear mechanistic interpretation of the combined osteolathyritic effects of the ßAPN:DTC data presented here is problematic. Although the mixture-response curves were consistent with dose-addition, strong correlation (p 0.90) with dose-addition was obtained for only two of the seven mixture-response curves. These results, coupled with significantly different slopes for the dose-response curves of ßAPN alone and DTC alone and the putative biphasic osteolathyritic response elicited by DTC alone, are considered important indications of a different dose-effect relationship due to differences in the action of the chemicals under study. Such results might be explained by actions of the chemicals that are different with respect to their molecular sites or modes of action but may share a common pathway; in essence similar to the "two-receptor one-transducer" model (Leff, 1987
; Scaramellini et al., 1997
). Therefore, the hypothesis presented here to explain the results is that the apparent biphasic nature of the DTC-alone response curve actually represents a second osteolathyritic effect of DTC (and that differences in lesion location should be systematically evaluated as the most likely source of the biphasic response). In such a multiple-mechanism scenario, the additional effect of a second mechanism might enhance the experimental mixture responses enough over the theoretical response calculated for independence to produce a combined effect equivalent to dose-addition.
Whether this hypothesis is subsequently confirmed or not, the results hold some important implications for evaluating chemical-mixture toxicity using this experimental design. The theory for a dose-additive combined effect assumes only one molecular site of action for a chemical. If a chemical such as DTC actually induces osteolathyrism in two ways within the same series of concentrations, the current theoretical models of chemical mixture toxicity would likely be inadequate, necessitating further model development. Even if this is so, data resulting from the current experimental design is amenable to other statistical approaches, such as response surface analysis, and, therefore, when evaluated over numerous data sets, has the potential to advance understanding of chemical mixture toxicity.
Although additional work needs to be done to determine the exact nature of ßAPN- and DTC-induced osteolathyrism and how it relates to the combined effect obtained herein, the results of this study are important for several reasons. First, the osteolathyritic effects of both ßAPN and DTC on Xenopus development are copper sensitive (although perhaps not in the same way), implicating copper as important in the osteolathyrogenic mechanism of these chemicals and providing direction for further mechanistic work at the biochemical level. Second, a combined effect statistically consistent with dose-addition was obtained despite ßAPN and DTC having statistically different slopes for their individual concentration-response curves, pointing out the potential importance of considering other factors such as slope and shape of the response curve in assessing the combined effect obtained using this approach. Third, the experimental design produces multiple mixture-response curves, providing a substantial amount of information to use in assessing chemical mixture toxicity.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Fax: (419) 289-5333. E-mail: ddawson2{at}ashland.edu.
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