Toxicology 147 (2000) 193–207 www.elsevier.com/locate/toxicol

Comparative evaluation of the combined osteolathyritic effects of two nitrile combinations on Xenopus embryos

Douglas A. Dawson a,*, Melissa A. Cotter a, Deidre L. Policz a, Deborah A. Stoffer a, Jason P. Nichols a, Gerald Po¨ch b

a Department of Biology/Toxicology, Ashland Uni6ersity, 401 College A6enue, Ashland, OH 44805, USA b Institute of Pharmacology and Toxicology, Uni6ersity of Graz, Graz, Austria

Received 30 November 1999; accepted 17 March 2000

Abstract

Two nitrile combinations, b- (bAPN) with aminoacetonitrile (AAN) and bAPN with bAPN (as a sham combination), were evaluated using the frog embryo mixture toxicity assay to determine their combined osteolathyritic effects and to compare the results with theoretical effects for two combined effects models. In separate tests each nitrile was tested with copper sulfate to determine the importance of copper in osteolathyrogen-induced disruption of cross-linking. Frog embryos (Xenopus lae6is) were exposed for 96 h, with daily solution removal and replacement. Preserved tadpoles were evaluated for osteolathyritic lesions. For the nitrile:nitrile combinations, the x2 goodness-of-fit test was used to compare the resulting mixture-response curves to theoretical curves for dose-addition and independence. For bAPN with AAN, the combined osteolathyritic effect for five of the seven mixture curves generated was greater than expected for each of the combined effects models. For bAPN with bAPN, the combined effect for all seven mixture curves was consistent with dose-addition, the combined effect expected for chemicals inducing toxicity by the same mechanism. For the nitrile:copper combinations, the EC50 for bAPN-induced osteolathyrism was increased two- to threefold (i.e. made less toxic) by co-administration with copper sulfate, while the EC50 for AAN-induced osteolathyrism was unchanged. The results are consistent with the idea that bAPN and AAN induce osteolathyrism, at least in part, by different mechanisms. © 2000 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: b-Aminopropionitrile; Aminoacetonitrile; Copper sulfate; Dose-addition; Independence; Chemical mixture toxicity

1. Introduction

 This research was conducted in compliance with the Ani- Osteolathyrism is defined as the failure of devel- mal Welfare Act and other federal statutes and regulations oping connective tissue fibers to cross-link prop- relating to animals and experiments involving animals and erly (Selye, 1957). Developing and elastin adhered to principles stated in the Guide for Care and Use of Laboratory Animals, NIH Publication 86-23 (1985 ed.). fibers are cross-linked using to oxi- * Corresponding author. dize the amino side group of peptidyl

0300-483X/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S0300-483X(00)00196-7 194 D.A. Dawson et al. / Toxicology 147 (2000) 193–207 residues within collagen and elastin into peptidyl affects proper connective tissue cross-linking. a-aminoadipic-d-semialdehyde (Pinnell and Mar- With the complexity of the cross-linking pro- tin, 1968). These can then form the cess, there are several opportunities for chemical covalent cross-linkages found in collagen and insult to be disruptive. As a result, osteolathyrism elastin fibers by condensing with amino groups or might occur through more than one mechanism. other peptidyl aldehydes (Kagan, 1986). Lysyl Some of the mechanisms that have been proposed oxidase (LO) is a copper-requiring that for osteolathyrism include: direct binding of the uses lysyltyrosine quinone as a (Wang et agent to LO (Tang et al., 1983), copper chelation al., 1996). Inhibition of LO activity adversely (Dasler and Stoner, 1959), blockage

Fig. 1. Frog embryo mixture toxicity assay design with 36 treatments. The design gives seven mixture-response curves: two with the concentration of chemical ‘A’ held at a fixed value with increasing concentrations of chemical ‘B’ (shaded columns), two vice versa

(shaded rows) and three fixed-ratio curves in which the concentrations of both chemicals increase (diagonal lines). The -fold EC50 values are based on a 1.2 factor, with 1.0 representing the EC50 for osteolathyrism for each chemical when tested alone. D.A. Dawson et al. / Toxicology 147 (2000) 193–207 195

Table 1 Concentration-osteolathyrism data for frog embryos exposed to chemical ‘A’ alone and chemical ‘B’ alone for tests of bAPN:AAN and bAPN:bAPN

Treatment no. bAPN:AAN bAPN:bAPN

Conc.a No. affectedb %c Conc.a No. affectedb %c

Chemical ‘A’ alone 20.028 7 14 0.024 3 6d 3150.041 30 0.035 6 13e 4 0.05820 40 0.050 15 30 50.070 32 64 0.060 27 55d 6 0.08434 72f 0.072 35 70 7 0.12149 98 0.104 49 98 Chemical ‘B’ alone 8 0.24 6 12 0.024 1 2d 9 0.3512 25d 0.035 7 14d 10 0.50 19 38 0.050 25 51d 11 0.6030 60 0.060 33 67d 120.72 38 76 0.072 36 72 13 1.0447 94 0.104 48 96

a mg/l-formula weight corrected. b Osteolathyritic. c For 50 survivors unless noted. d 49 survivors. e 48 survivors. f 47 survivors.

(Levene, 1971), oxidative stress (Ghate, 1985), attributed to bAPN (Levene and Carrington, and disruption of the cofactor (Bird and Levene, 1986). In addition, quantitative structure-activity 1982; Kagan and Trackman, 1991). relationship (QSAR) studies did not develop a Osteolathyrism can be induced by a variety of single high-quality QSAR equation for oste- chemicals, including: nitriles, ureides, hydrazides, olathyrism induced by various acid hydrazides, hydrazines (Levene, 1961), alkyl carbazates (Daw- thiosemicarbazides, benzoic acid hydrazides, and son et al., 1991) and dithiocarbamates (Bancroft alkyl carbazates (Schultz and Ranney, 1988; Daw- and Prahlad, 1973). Among the nitriles capable of son et al., 1990, 1991), suggesting that more than inducing osteolathyrism are b-aminopropionitrile one mechanism of osteolathyrism exists among (bAPN), aminoacetonitrile (AAN) and methylene the various chemical classes. aminoacetonitrile (Levene, 1961). The mechanism Frog embryos, including Xenopus lae6is (Ban- for bAPN-induced osteolathyrism is reported to croft and Prahlad, 1973) and Microhyla ornata be direct binding of the agent to the enzyme, as (Ghate and Mulherkar, 1980), are susceptible to the compound has been shown to covalently bind chemically-induced osteolathyrism. Primary oste- LO, with the amount of inactivation proportional olathyritic effects include, at lower concentra- to the amount of bAPN bound (Tang et al., tions, small dorso-ventral bands of 1983). Whether this is the general mechanism of disorganization within the notochord or an osteolathyrism for some of the other osteolathyro- outpocketing of the ventral margin of the noto- gens, however, is not clear. For example, work chord. These initial lesions are clearly observed with the chick indicated that (a within the notochord of the transparent tadpoles ureide) may have a mechanism of osteolathyritic using a dissecting microscope. At higher oste- action that is different from the one that has been olathyrogen concentrations extensive disorganiza- 196 D.A. Dawson et al. / Toxicology 147 (2000) 193–207 tion, enlargement and/or folding of the notochord the chemicals induce toxicity by different mecha- and disruption of the notochordal sheath are ob- nisms (Po¨ch et al., 1996). Initial evaluations of the served, leading, at the gross level, to a ‘wavy-tail’ approach (Dawson and Po¨ch, 1997; Mentzer et appearance (Schultz et al., 1985; Dawson et al., al., 1999) have examined osteolathyrogen combi- 1990). Notochord effects have been further char- nations because of the potential for different acterized using electron microscopy to include mechanisms of action among the different chemi- disorganization of collagen fibers and, in some cal classes. With this possibility it was hypothe- instances, absence of the elastic externa (Schultz sized that osteolathyrism would provide a et al., 1985; Riggin and Schultz, 1986). rigorous test of the toxicity and statistical The frog embryo mixture toxicity assay (Daw- methodologies developed and determine if the son and Po¨ch, 1997), a derivation of FETAX approach is sensitive enough to detect variations (Dumont et al., 1983), was developed to deter- in the resulting combined effects that may be mine the toxicity of binary chemical mixtures and indicative of chemicals acting by different specific to attempt to relate that toxicity to common or mechanisms (i.e. direct enzyme binding, copper different mechanisms of chemical action. This is chelation, cofactor disruption, etc.). Identification done by statistically comparing the experimental of such variations would provide direction for results with theoretical effects for each of two examining potential mechanistic differences at the combined effects models: dose-addition and inde- biochemical level. As a part of this project, which pendence. A dose-addition combined effect is ex- will examine many osteolathyrogen combinations pected when two chemicals induce toxicity by the across several chemical classes, two nitrile:nitrile same mechanism of action, while a combined combinations: bAPN with AAN and bAPN with effect consistent with independence indicates that bAPN were tested. To determine the importance

Table 2 Concentration-osteolathyrism data for frog embryos exposed to the fixed AAN concentrations with increasing bAPN concentra- tions, expected numbers for dose-addition and independence models of combined effects, and data fit to the models

Treatment no. [bAPN]a No. osteolathyritic No.b expected if dose-addition No.b expected if independence

[AAN]a =0.35 mg/l per treatment 140.014 25 22 13 19c 0.028 38 30 15 23c 0.041 47 36 20 29c 0.058 48 41 29 330.08450 46 40 340.12150 48 47 x2 =8.2 x2 =101.1 d.f.=5 d.f.=5 P\0.10 PB0.0005 [AAN]a =0.5 mg/l per treatment 150.01442 26 18 170.020 45 30 18 24 0.041 50 39 24 30c 0.058 49 42 31 36 0.175 50 49 49 x2 =22.1 x2 =112.9 d.f.=4 d.f.=4 PB0.0005 PB0.0005

a mg/l-formula weight corrected. b Rounded c 49 survivors, all other treatments had 50. D.A. Dawson et al. / Toxicology 147 (2000) 193–207 197

Table 3 Concentration-osteolathyrism data for frog embryos exposed to the fixed bAPN concentrations with increasing AAN concentra- tions, expected numbers for dose-addition and independence models of combined effects, and data fit to the models

Treatment no.[AAN]a No. osteolathyritic No.b expected if dose-addition No.b expected if independence

[iAPN]a =0.041 mg/l per treatment 21 0.1234 25 16 22 0.24 38 34 17 23c 0.3547 38 20 240.5050 44 30 25 0.7250 47 40 26 1.04 50 49 49 x2 =7.3 x2 =102.0 d.f.=5 d.f.=5 P=0.20 PB0.0005 [iAPN]a =0.058 mg/l per treatment 27 0.1240 29 21 280.17 46 33 21 29c 0.3548 40 25 30c 0.50 49 43 31 31 1.550 50 49 x2 =12.0 x2 =80.8 d.f.=4 d.f.=4 PB0.025 PB0.0005

a mg/l-formula weight corrected. b Rounded. c 49 survivors, all other treatments had 50. of copper in osteolathyrism-induction, both feeding days and, at minimum, 6 days per week. bAPN and AAN were also tested with copper Frogs to be bred were not fed on the day of sulfate. breeding to limit food regurgitation in the breed- ing tank. FETAX solution was used in the breed- ing tank and for all embryo exposures prior to 2. Methods fixation (Dawson and Bantle, 1987). Frog breed- ing was induced by aqueous injections of human 2.1. Adult frog care chorionic gonadotropin (Sigma, St. Louis, MO) into the dorsal lymph sac of the adults. Addi- Adult X. lae6is (Xenopus I, Dexter, MI) upon tional details of Xenopus care and breeding proce- arrival in the lab, were acclimated to standard dures can be found elsewhere (Bantle et al., 1990; housing conditions for 1 week prior to prophy- ASTM, 1991; Dawson et al., 1992). lactic treatment with ivermectin (Dawson et al., 1992). This procedure greatly reduces the fre- 2.2. Assay procedures quency of outbreaks of a nematode-induced skin infection. After breeding, collected embryos were placed Adult frogs, used for breeding purposes only, ina2%(w/v) cysteine solution (pH 8.1) and were housed in 10-gal glass aquaria in 3–4 gal of gently swirled for 1–2 min, to remove the jelly filtered tap water. The frogs were fed a mixture of coat around the embryos. Embryos were then raw, ground beef liver and frog brittle (Nasco, Ft. rinsed several times in FETAX solution. Obvi- Atkinson, WI) three to five times per week. ously dead, necrotic and unfertilized embryos Aquaria were cleaned immediately thereafter on were discarded. The remaining embryos were ex- 198 D.A. Dawson et al. / Toxicology 147 (2000) 193–207 amined under a dissecting microscope and those separate stock solutions of bAPN were prepared judged to be developing normally at blastula just as if two different chemicals were being through early gastrula stages were retained for tested). The 36 treatments were then made from testing. these stock solutions. The treatments were se- The osteolathyrogens used in testing were b- lected using a 1.2-factor matrix design (Dawson aminopropionitrile monofumarate salt (CAS c and Po¨ch, 1997), resulting in two single-chemical 2079-89-2; Sigma) and aminoacetonitrile bisulfate and seven mixture concentration-response curves (CAS c 151-63-3; Aldrich, Milwaukee, WI). Pre- (Fig. 1). The latter includes two curves in which vious work with these osteolathyrogens had estab- the chemical ‘A’ concentration was held at a fixed lished the concentrations of each chemical alone value with increasing concentrations of chemical that caused 50% of exposed embryos to develop ‘B’, two vice versa, and three fixed-ratio curves in osteolathyritic lesions (i.e. EC50 osteolathyrism). which the concentration of both chemicals in- Stock solutions of each osteolathyrogen were pre- creased. All reported osteolathyrogen concentra- pared daily (for the bAPN:bAPN combination, tions have been formula-weight corrected.

Table 4 Concentration-osteolathyrism data for frog embryos exposed to increasing concentrations of bAPN and AAN at 1:1, 1:3 and 3:1 effect ratios, expected numbers for the dose-addition and independence models of combined effects and data fit to the models

Treatment no. [bAPN]a [AAN]a No. No.b expected if dose-addition No.b expected if independence osteolathyritic

1:1 160.0160.14 12 8 4 180.0280.24 33 21 8 23c 0.041 0.35 47 35 17 30c 0.058 0.50 49 44 34 35 0.121 1.04 50 50 50 x2 =14.6 x2 =161.6 d.f.=4 d.f.=4 PB0.01 PB0.0001 1:3 14 0.014 0.35 25 14 7 17 0.020 0.5045 33 20 200.028 0.72 50 44 36 260.041 1.04 50 48 45 31 0.058 1.50 50 49 49 x2 =14.6 x2 =83.5 d.f.=4 d.f.=4 PB0.01 PB0.0005 3:1 21 0.041 0.12 34 21 13 28 0.058 0.17 46 35 24 320.0840.24 50 45 38 340.121 0.35 50 49 46 36 0.175 0.50 50 50 49 x2 =13.5 x2 =62.3 d.f.=4 d.f.=4 PB0.01 PB0.0005

a mg/l-formula weight corrected. b Rounded. c 49 survivors, all other treatments had 50. D.A. Dawson et al. / Toxicology 147 (2000) 193–207 199

each of those three copper sulfate concentrations with seven osteolathyrogen concentrations (i.e. 21 treatments). Two separate tests of bAPN with copper were conducted and the results combined, giving a total of 80 embryos per treatment. The AAN:copper test had 60 embryos per treatment (three dishes of 20 embryo/dish). Following exposure, preserved (1% formalin, pH 7.0) tadpoles were microscopically examined for osteolathyritic lesions and for any other mal- formations present, using an atlas of frog malfor- mations as a guide (Bantle et al., 1990). The Fig. 2. Concentration-osteolathyrism curve for bAPN alone (open circles), AAN alone (open squares) and their 1:1 mixture non-osteolathyritic malformations were recorded (closed circles). Theoretical effects calculated for the dose-ad- in case a combination produced a malformation dition (dashed line) and independence (dotted line) models of not observed upon exposure to either chemical combined effect are included. alone or a concentration-response relationship for these malformations became evident within a given mixture.

2.3. 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 (P50.05) in rates Fig. 3. Concentration-osteolathyrism curve of AAN alone of non-osteolathyritic malformations between (open circles) and in the presence of 0.058 mg/l bAPN (closed each treatment and the control and across all squares), along with the calculated frequency curve for the treatments, respectively. dose-addition (dashed line) model of combined effect. The dash-dot line depicts the biphasic nature of the concentration- response curve for AAN tested alone.

Embryos were exposed in covered, glass Petri dishes containing 10 ml of solution for 96 h at 23.5°C. There was daily removal and replacement of solutions, with any dead embryos removed from the dishes. For the nitrile:nitrile tests, each treatment had 50 embryos divided equally be- tween two dishes. For the nitrile:copper tests, cupric sulfate-pen- tahydrate (CAS c 7758-99-8; Fisher Scientific) was dissolved in FETAX solution at a 500-ml Fig. 4. Concentration-osteolathyrism curve of bAPN alone volume and this stock solution was used each day (open circles) and in the presence of 0.347 mg/l AAN (closed of the 4-day tests. These tests had 32 treatments: circles), along with the calculated frequency curve for the dose-addition (dashed line) model of combined effect. The one control, seven osteolathyrogen-only, three dash-dot line depicts the biphasic nature of the concentration- copper sulfate-only (0.5, 1.0 and 1.5 mg/l), and response curve for bAPN tested alone. 200 D.A. Dawson et al. / Toxicology 147 (2000) 193–207

Table 5 Concentration-osteolathyrism data for frog embryos exposed to fixed concentrations of bAPN with increasing bAPN concentra- tions, expected numbers for dose-addition and data fit to the model

Fixed-dose of bAPN as chemical ‘B’ Fixed-dose of bAPN as chemical ‘A’

[bAPN]a No.No.b expected if [bAPN]a No. No.b expected if osteolathyriticdose-addition osteolathyritic dose-addition

[‘B’]=0.035 mg/la per treatmentc [‘A’]=0.035 mg/la per treatmentd 0.012 16 17 0.012 13e 18 0.024 2830 0.024 26f 29 0.0354138 0.035 41 37 0.050 4344 0.050 46 43 0.072 48 48 0.072 50 47 0.104 4950 0.104 50 49 x2 =0.5 x2 =2.43 d.f.=5 d.f.=5 P\0.99 P\0.70 [‘B’]=0.050 mg/la per treatmentg [‘A’]=0.050 mg/la per treatmenth 0.012 30 35 0.012 27 26 0.017 4139 0.017 38f 30 0.035 46 45 0.035 43 41 0.050 4848 0.050 48 45 0.15048e 48 0.150 50 50 x2 =0.9 x2 =2.43 d.f.=4 d.f.=4.0 P\0.90 P\0.60

a mg/l-formula weight corrected. b Rounded. c Treatment numbers were 14, 19, 23, 29, 33, 34. d Treatment numbers were 21–26. e 48 survivors, all other treatments except ‘f’ (below) had 50. f 49 survivors, all other treatments except ‘e’ (above) had 50. g Treatment numbers were 15, 17, 24, 30, 36. h Treatment numbers were 27–31.

For the nitrile:nitrile combinations, experimen- concentration-response relationships for such tal data points for osteolathyrism induced by malformations were produced. chemical ‘A’ alone and by chemical ‘B’ alone were fit to sigmoidal curves using a four parameter 2.3.1. Theoretical dose-addition cur6e calculation logistic function along with worksheets and data for comparison with fixed-dose responses program files designed (Po¨ch, 1993; Po¨ch et al., These calculations were based on the principle 1997) for use with SigmaPlot (Jandel Scientific, described by Po¨ch et al. (1990). This procedure San Rafael, CA). Curve fit parameters were mini- was conducted using SigmaPlot as detailed by mum effect (a), slope (b), EC50 osteolathyrism (c), Holzmann et al. (1999). In essence the theoretical and maximum effect (d). Then, for each experi- curve is obtained by calculating dose x of chemi- mental response curve, theoretical curves for the cal ‘A’ with which the concurrently applied fixed- number of osteolathyritic tadpoles were calculated dose of chemical ‘B’ was equieffective, using the for the dose-addition and independence models as equation: x={([(a−d)/(a2−d)]−1)(c b)}1/b detailed below. Non-osteolathyritic malforma- where a–d are the parameters (defined above) for tions were not included in these analyses since no the curve of chemical ‘A’ alone and a2isthe D.A. Dawson et al. / Toxicology 147 (2000) 193–207 201

minimum effect for the curve of chemical ‘A’ with EC50A) with a common slope of ‘A’ and ‘B’ the concurrently applied fixed-dose of chemical calculated as (B slope+A slope)/2. The EC50 of ‘B’. From the definition of dose-addition, the the dose-addition curve=1/leftshift×EC50 of the effects of chemical ‘A’ alone are to be expected at more effective agent. the concentrations of A−x. Therefore, the re- sponse values for the theoretical dose-addition curves of chemical ‘A’ correspond to simulated 2.3.3. Theoretical independence cur6e calculations concentrations of A−x. These were calculated based on the effects E of

‘A’ and ‘B’ alone: EA + B =EA +EB −(EAEB) 2.3.2. Theoretical dose-addition cur6e calculation where E is expressed as the fraction of the maxi- for comparison with fixed-ratio responses mum response and was corrected for oste- The leftshift from the concentration-oste- olathyrism in controls (x) using the equation olathyrism curve of chemical ‘B’=1+(EC50B/ E=(E−x)/(1−x).

Table 6 Concentration-osteolathyrism data for frog embryos exposed to increasing concentrations of bAPN and bAPN at 1:1, 1:3, and 3:1 effect ratios, expected numbers for dose-addition and data fit to the models

Treatment no.[bAPN]a [bAPN]a No. osteolathyritic No.b expected if dose-addition

1:1 16 0.014 0.014 8 4 18 0.024 0.024 16 17 23 0.035 0.035 41 37 300.050 0.050 48 47 35 0.104 0.104 50 50 x2 =4.5 d.f.=4 P\0.40 1:3 14 0.012 0.035 25c 18 17 0.0170.050 41 38 20 0.0240.072 41d 45 260.035 0.104 50 50 31 0.050 0.150 50 50 x2 =3.7 d.f.=4 P\0.40 3:1 21 0.035 0.012 13d 13 28 0.050 0.01738c 33 32 0.072 0.024 44 46 34 0.104 0.035 49 49 360.1500.050 48d 48 x2 =0.95 d.f.=4 P\0.95

a mg/l-formula weight corrected. b Rounded. c 49 survivors, all others 50, except ‘d’ (below). d 48 survivors, all others 50, except ‘c’ (above). 202 D.A. Dawson et al. / Toxicology 147 (2000) 193–207

Table 7 Concentration-osteolathyrism data for frog embryos exposed to various fixed concentrations of copper sulfate with increas- ing concentrations of bAPN or AAN

b APN:CuSO4 AAN:CuSO4

[bAPN]a % [AAN]a % Osteolathyriticc Osteolathyriticb

[CuSO4]=0 mg/l 0001d e Fig. 5. Concentration-osteolathyrism curve of bAPN alone 0.020 0 0.24 2 0.035 1120.42 (open circles) and in the presence of 0.0347 mg/l bAPN (closed circles), along with the calculated frequency curve for the 0.0506 0.60 17 0.060 11f 0.72 23 dose-addition (dashed line) and independence (dotted line) f models of combined effect. The mixture curve exemplifies the 0.072 24 0.86 37 0.10463f 1.25 75 best fit of experimental mixture toxicity data to the dose-addi- e tion model. 0.180 96 2.15 92

[CuSO4]=0.5 mg/l 2.3.4. Fixed-ratio response calculations 0012 d These calculations required that chemical ‘B’ 0.035 1 0.42 15 0.0601e 0.72 23 concentrations be converted to equivalent concen- 0.104 6e 1.25 70 trations of chemical ‘A’ for each fixed-ratio (i.e. 0.15019 1.80 85 1:3, 1:1, 3:1) using the equation B×factor A/B 0.216 54e 2.59 90 (Po¨ch et al., 1997). 0.373 88d 4.48 98g d The x2 goodness-of-fit test was used to compare 0.645 96 7.74 100 the experimental responses with the theoretical [CuSO4]=1.0 mg/l d responses (Po¨ch, 1993). The analyses were used to 00 00 0.035 3f 0.42 13 determine whether the combined osteolathyritic 0.0604d 0.72 25 effects differed significantly (P50.05) from the 0.104 4d 1.25 73 theoretical effects for dose-addition and for 0.1509f 1.80 80 independence. 0.216 27e 2.59 93 e Octanol/water partition coefficient values for 0.37374 4.48 98 0.645 94f 7.74 100 bAPN and AAN were either calculated or re- trieved as measured values using CLogP for Win- [CuSO4]=1.5 mg/l d dows software (BIOBYTE, Claremont, CA). 001 0 0.035 1f 0.42 20 0.060 3220.72 0.1041e 1.25 70 0.1501d 1.80 83 3. Results 0.216 28h 2.59 95 0.37368d 4.48 98 i 3.1. iAPN:AAN combination 0.645 90 7.74 100 a mg/l-formula weight corrected. In the control treatment, there were no deaths b 80 embryos exposed. and no non-osteolathyritic malformations, but c 60 embryos exposed. there was one tadpole (2.0%) with an oste- d 77 survivors. e olathyritic lesion. 78 survivors. f 79 survivors. Across all treatments the incidences of death g 59 survivors. and non-osteolathyritic malformations were 0.4 h 76 survivors. and 2.2%, respectively. Non-osteolathyritic mal- i 75 survivors. D.A. Dawson et al. / Toxicology 147 (2000) 193–207 203

sponses were not significantly different from dose- addition (both the lower of the two ‘fixed-dose’ curves for each chemical), the fit with the model was not high (i.e. 0.10BPB0.20 (Table 2) and P=0.20 (Table 3, Fig. 4)).

3.2. iAPN:iAPN combination

In the control treatment, there was one death (2.0%) and one osteolathyritic tadpole (2.0%). Three tadpoles (6.1%), including the one with osteolathyrism, had non-osteolathyritic malfor- mations, typified by gut and/or head and eye defects. Across all treatments the incidences of death and non-osteolathyritic malformations were 0.9 and 4.9%, respectively. Non-osteolathyritic mal- formations included gut (4.6%), edema (3.8%), eye (3.2%), head (2.7%), tail (2.2%) and fin (0.2%) defects, with multiple defects in 74 of 88 tadpoles having non-osteolathyritic malformations. No Fig. 6. Representative concentration-osteolathyrism curves for treatment had more than six animals with non-os- (a) bAPN-alone (open circles) and with 1.0 mg/l copper sulfate teolathyritic malformations. There was no statisti- (closed squares) and for (b) AAN-alone (open circles) and with cal association between non-osteolathyritic defects 1.0 mg/l copper sulfate (closed squares). and chemical exposure. Again, the osteolathyritic lesions observed in the treatments were typical of formations included gut (1.7%), eye (1.5%), edema the initial lesions described previously (Section 1). (1.0%), head (0.8%), tail (0.3%), and fin (0.1%) Concentration-osteolathyrism data for bAPN defects, with multiple defects in 30 of the 39 alone as chemical ‘A’ and for bAPN alone as tadpoles having non-osteolathyritic malforma- chemical ‘B’ (Table 1) were used to calculate tions. No treatment had more than four tadpoles theoretical effects for the dose-addition model of with non-osteolathyritic malformations. There combined effect. The incidences of osteolathyrism was no statistical association between non-oste- for each of the seven experimental mixture-re- olathyritic malformations and chemical exposure. sponse curves were not statistically different from Osteolathyritic lesions observed in the exposure dose-addition, with P\0.40 to P\0.99 (Tables 5 treatments were the initial lesions described in and 6, Fig. 5). All seven experimental curves had Section 1. a combined effect greater than expected (PB Concentration-osteolathyrism data for bAPN 0.0005) for independence (data not shown). alone and AAN alone (Table 1) were used to calculate theoretical effects for each of the com- 3.3. Copper tests bined effects models. The incidences of oste- olathyrism for each of the seven experimental For the bAPN:copper tests, three controls died mixture-response curves were significantly greater (3.8%), three had non-osteolathyritic malforma- than expected for the independence model (PB tions (3.9%) and there was one osteolathyritic 0.0005), and were significantly greater than ex- tadpole (1.3%). Death for the three copper-only pected for the dose-addition model (PB0.025) for treatments was 2.5%, with 3.0% of survivors hav- five of the seven mixture curves (Tables 2–4, Figs. ing non-osteolathyritic defects (generally of the 2 and 3). For the two curves in which the re- gut, eye, and face), and 1.3% with osteolathyritic 204 D.A. Dawson et al. / Toxicology 147 (2000) 193–207

3 lesions. Across all treatments there was 2.4% for treatment 3 is equal to its EC50/1.2 (i.e. death and a 5.6% incidence of non-osteolathyritic EC50 ×0.58), while the concentration for treat- 3 malformations. There were no statistical differ- ment 7 is EC50 ×1.2 (i.e. EC50 ×1.73). The 23 ences in the incidences of death or non-oste- mixture treatments are then based on this scheme olathyritic malformations between each treatment and result in seven mixture-response curves. The and the control or when each treatment was com- 1.2-factor design is useful for chemicals with a pared with all other treatments. Osteolathyritic toxicity slope between 2 and 8. Other factors defects were as described above. Co-administra- could be used for chemicals with toxicity slopes tion of copper sulfate at 0.5, 1.0 and 1.5 mg/l outside this range (Dawson and Po¨ch, 1997). b increased the EC50 for APN-induced oste- Coupled with the experimental design is the use olathyrism from 0.093 mg/l bAPN-alone to 0.207, of combined effects models with which to com- 0.279 and 0.281 mg/l, respectively (Table 7). pare the experimental responses. The dose-addi- For the AAN:copper test, there were no control tion and independence models are used because or copper-only deaths, a 4.6% incidence of non- they theoretically relate to events at the molecular osteolathyritic malformations (gut, eye, face, sites of action (Po¨ch et al., 1996). For dose-addi- edema, fin), and a 0.4% incidence of oste- tion, the agents are presumed to share a single olathyrism. Across all treatments there was one molecular site of action because one chemical acts death (0.05%) and a 4.9% incidence of non-oste- as a dilution of the other and they share a com- olathyritic defects (gut, eye, face, edema, fin). mon dose-response curve slope. For independence There were no statistical differences in the inci- (Bliss, 1939), the chemicals act at different sites dences of death or non-osteolathyritic malforma- and the toxicity of one does not affect the toxicity tions between each treatment and the control or of the other. Therefore, an independent combined across all treatments. Osteolathyritic lesions were effect indicates that the chemicals are likely to as described above, except for the highest AAN work by different mechanisms. Other combined concentration in which the more severe ‘wavy-tail’ effects models can also be used to compare exper- lesions were also observed. Co-administration of imental with theoretical results, but mechanistic copper sulfate had no effect on the EC50 for assessments of such comparisons may be inappro- AAN-induced osteolathyrism with these values priate (Po¨ch et al., 1996). being 0.97, 1.02, 0.98 and 1.04 mg/l for AAN- alone and with 0.5, 1.0 and 1.5 mg/l copper 4.1. Combination effects sulfate, respectively (Table 7, Fig. 6). The combined effects obtained for the ni- trile:nitrile and nitrile:copper tests show several 4. Discussion differences. The bAPN:AAN combination pri- marily produced toxicity exceeding dose-addition, The 1.2-factor mixture toxicity testing design while the bAPN:bAPN combination showed toxi- employed in this study represents a synthesis of city consistent with dose-addition. Addition of the fixed-dose (Po¨ch, 1993) and fixed-ratio (Daw- copper sulfate increased the amount of bAPN son, 1994) testing strategies. In the current design needed to induce osteolathyrism in 50% of the each chemical is tested alone at six concentra- frog embryos, by two to three-fold. Addition of tions, treatments 2–7 for chemical ‘A’ and 8–13 copper sulfate, however, had no effect on oste- for chemical ‘B’. These treatments were selected olathyrism induced by the concentrations of AAN by assigning the EC50 for osteolathyrism induced tested. by each chemical alone the value 1.0 (treatments 5 Based on similarity of chemical structures b   and 11 for chemicals ‘A’ and ‘B’, respectively). ( APN-NH2CH2CH2C N; AAN-NH2CH2C N), The other five single treatments/chemical are de- these differences would not be expected. The re- termined by multiplying or dividing the EC50 by sult might initially be explained in a number of 1.2x. For example, the chemical ‘A’ concentration ways: (1) the presence of one chemical increased D.A. Dawson et al. / Toxicology 147 (2000) 193–207 205 uptake of the second, thereby increasing toxicity zyme (Smith-Mungo and Kagan, 1998). Without over that expected for two chemicals working by this information, it has not been determined ex- the same mechanism, (2) one or both of the actly how bAPN inactivates the enzyme. If chemicals causes osteolathyrism in more than bAPN were to compete with or otherwise affect one way, somehow combining to increase toxic- copper in LO and inactivate the enzyme as a ity, or (3) the chemicals work, at least in part, result, and if copper were also important in co- by different mechanisms, causing greater than factor regeneration or function, bAPN could af- expected toxicity. fect proper connective tissue cross-linking at two The first explanation appears unlikely as the points in the process. As noted earlier, LO does log 1-octanol/water partition coefficient values require copper for proper enzyme function (Ka- for these chemicals are similar at −1.06 for gan and Trackman, 1991) and it is likely that bAPN (modeled) and −1.37 for AAN (mea- regeneration of the lysyltyrosine quinone cofac- sured). tor also requires copper (Wang et al., 1996; The second possibility, that one (or both) of Smith-Mungo and Kagan, 1998; Dooley, 1999), the chemicals induces osteolathyrism in more since copper has been shown to be necessary for than one way, may have some merit. A close other similarly functioning oxidase cofac- examination of the concentration-response tors (Janes et al., 1992; Matsuzaki et al., 1994). curves for AAN alone (Fig. 3, open circles) and Although these ideas provide support for the bAPN alone (Fig. 4, open circles) suggests the idea of multiple osteolathyritic mechanisms for a possibility of biphasic curves, a potential indica- single chemical they do not, alone, explain why tor of two osteolathyritic effects. While this pos- excess toxicity was observed for most sibility must be examined further, there are at bAPN:AAN mixture-response curves when the least two possibilities for multiple osteolathyritic sham combination showed only dose-addition. mechanisms that can be suggested from the lit- Therefore, the nitrile:nitrile and nitrile:copper re- erature. First, in addition to being needed in the sults obtained suggest that bAPN and AAN in- extracellular matrix for normal cross-linking, ly- duce osteolathyrism, at least in part, by different syl oxidase (LO) has been shown to be active mechanisms, one involving copper for bAPN within the nuclei of fibrogenic cells (Li et al., and one not involving copper for AAN. While 1997). While the biologic role of nuclear LO has further work on these possibilities is needed, the not been fully determined, it has been reported assay and statistical methods described herein, to affect chromatin organization (Mello et al., appear to have provided direction for research 1995). It was proposed, therefore, that nuclear at the biochemical level aimed at discerning the LO might affect regulatory events within nuclei mechanisms. In addition, the approach appears (Li et al., 1997). If this led to impaired connec- to have value in improving understanding of the tive tissue development, as happens when LO complexities of chemical mixture toxicity at the activity is inhibited in the extracellular matrix, a organismal level. single osteolathyrogen would have the potential to induce osteolathyrism in two ways. A second possibility centers on the protective Acknowledgements effect of added copper on bAPN-induced oste- olathyrism. While bAPN has been shown to This publication was made possible by grant 1 cause osteolathyrism by directly binding to LO R15 ES08019-01 from the National Institute of (Tang et al., 1983), the proposed mechanism of Environmental Health Sciences (NIEHS), NIH. LO action in connective tissue fiber cross-linking Its contents are solely the responsibility of the has not been finalized due to difficulties in ob- authors and do not represent the official views taining a complete structural analysis of the en- of the NIEHS, NIH. 206 D.A. Dawson et al. / Toxicology 147 (2000) 193–207

References Dunnett, C.W., 1955. A multiple comparisons procedure for comparing several treatments with a control. J. Am. Stat. ASTM, 1991. Standard guide for conducting the frog embryo Assoc. 50, 1096–1121. teratogenesis assay: Xenopus (FETAX). In: Biological Ef- Ghate, H., 1985. Dithiocarbamate-induced teratogenesis in fects and Fate, E47-01-Aquatic Toxicology. American So- frog embryo. Riv. Biol. 78, 288–291. ciety for Testing and Materials, Philadelphia, PA Ghate, H.V., Mulherkar, L., 1980. Effect of sodium di- E1439-91. ethyldithiocarbamate on developing embryos of the frog Bancroft, R., Prahlad, K.V., 1973. Effect of ethylenebis(dithio- Microhyla ornata. Ind. J. Exp. Biol. 18, 1040–1042. carbamic acid) disodium salt (nabam) and ethylenebis(- Holzmann, S., Dittrich, P., Po¨ch, G., 1999. Dose-response dithiocarbamato)manganese (maneb) on Xenopus lae6is curves of vasorelaxants: computerized calculation of com- development. Teratology 7, 143–150. bination effects using the example of nicorandil. J. Clin. Bantle, J.A., Dumont, J.N., Finch, R.A., Linder, G., 1990. Basic Cardiol. 2, 96–98. Atlas of Abnormalities: A Guide for the Performance of Janes, S.M., Palcic, M.M., Scaman, C.H., Smith, A.J., Brown, FETAX. Oklahoma State University Publishing, Stillwa- D.E., Dooley, D.M., Mure, M., Klinman, J.P., 1992. Iden- ter, OK 69 pp. tification of topaquinone and its consensus sequence in Bird, T.A., Levene, C.I., 1982. Lysyl oxidase (EC1.4.3.13): copper amine oxidases. Biochemistry 31, 12147–12154. evidence that pyridoxal phosphate is a cofactor. Biochim. Kagan, H.M., 1986. Characterization and regulation of LO. Biophys. Res. Commun. 108, 1172–1180. In: Mecham, R.P. (Ed.), Regulation of Matrix Accumula- Bliss, C.I., 1939. The toxicity of poisons applied jointly. Ann. tion. In: Biology of the Extracellular Matrix, vol. 1. Aca- Appl. Biol. 26, 585–615. demic Press, Orlando, FL, pp. 321–398. Dasler, W., Stoner, R.G., 1959. Production of spinal curves in Kagan, H.M., Trackman, P.C., 1991. Properties and function rats by chemically dissimilar substances. Experentia 15, of lysyl oxidase. Am. J. Respir. Cell Mol. Biol. 5, 206–210. 112–115. Levene, C.I., 1961. Structural requirements for lathyrogenic Dawson, D.A., 1994. Chemical mixture toxicity assessment agents. J. Exp. Med. 114, 295–310. using an alternative-species model: applications, opportu- Levene, C.I., 1971. Effects of lathyrogenic compounds on the nities and perspectives. In: Yang, R.S.H. (Ed.), Toxicology cross-linking of collagen and elastin in vivo. In: Aldridge, of Chemical Mixtures: Case-Studies, Mechanisms, and W.N. (Ed.), Mechanisms of Toxicity. St. Martin’s Press, Novel Approaches. Academic Press, San Diego, pp. 539– New York, pp. 67–85. 563. Levene, C.I., Carrington, M.J., 1986. The inhibition of Dawson, D.A., Bantle, J.A., 1987. Development of a reconsti- -lysine 6-oxidase by various lathyrogens: evidence tuted water medium and preliminary validation of the frog for two different mechanisms. Biochem. J. 232, 293–296. embryo teratogenesis assay-Xenopus (FETAX). J. Appl. Li, W., Kaliappanadar, N., Strassmaier, T., Graham, L., Toxicol. 7, 237–244. Thomas, K.M., Kagan, H.M., 1997. Localization and ac- Dawson, D.A., Po¨ch, G., 1997. A method for evaluating tivity of lysyl oxidase within nuclei of fibrogenic cells. Proc. mechanistic aspects of chemical mixture toxicity. Toxicol. Natl. Acad. Sci. USA 94, 12817–12822. Methods 7, 267–278. Matsuzaki, R., Fukui, T., Sato, H., Ozaki, Y., Tanizawa, K., Dawson, D.A., Schultz, T.W., Baker, L.L., Mannar, A., 1990. 1994. Generation of the topa quinone cofactor in bacterial Structure-activity relationships for osteolathyrism: III. monoamine oxidase by cupric ion-dependent autooxida- Substituted thiosemicarbazides. J. Appl. Toxicol. 10, 59– tion of a specific tyrosyl residue. FEBS Lett. 351, 360–364. 64. Mello, M.L., Contente, S., Vidal, B.C., Planding, W., Schenck, Dawson, D.A., Schultz, T.W., Baker, L.L., 1991. Structure-ac- U., 1995. Modulation of ras transformation affecting chro- tivity relationships for osteolathyrism: IV. p-Substituted matin supraorganization as assessed by image analysis. benzoic acid hydrazides and alkyl carbazates. Environ. Exp. Cell Res. 220, 374–382. Toxicol. Chem. 10, 455–461. Mentzer, R.K., Smith, N.D., Po¨ch, G., Dawson, D.A., 1999. Dawson, D.A., Schultz, T.W., Schroeder, E.C., 1992. Labora- Combined osteolathyric effects of b-aminopropionitrile tory care and breeding of the African clawed frog. Lab. and penicillamine on Xenopus embryos: statistical compari- Anim. 21, 31–36. son with dose-addition and independence. Drug Chem. Dooley, D.M., 1999. Structure and biogenesis of topaquinone Toxicol. 22, 359–374. and related cofactors. J. Biol. Inorg. Chem. 4, 1–11. Pinnell, S.R., Martin, G.R., 1968. The cross-linking of colla- Dumont, J.N., Schultz, T.W., Buchanan, M., Kao, G., 1983. gen and elastin: enzymatic conversion of lysine in peptide Frog embryo teratogenesis assay: Xenopus (FETAX) — a linkage to a-aminoadipic-d-semialdehyde (allysine) by an short-term assay applicable to complex environmental mix- extract from . Proc. Natl. Acad. Sci. USA 61, 708– tures. In: Waters, M.D., Sandhu, S.S., Lewtas, J.E., Clax- 714. ton, L., Chernoff, N., Nesnow, S. (Eds.), Short-Term Po¨ch, G., 1993. Combined Effects of Drugs and Toxic Agents: Bioassays in the Analysis of Complex Environmental Mix- Modern Evaluation in Theory and Practice. Springer, Vi- tures III. Plenum, New York, pp. 393–405. enna 167 pp. D.A. Dawson et al. / Toxicology 147 (2000) 193–207 207

Po¨ch, G., Dawson, D.A., Dittrich, P., 1997. Teratogenic mix- Schultz, T.W., Dumont, J.N., Epler, R.G., 1985. The embry- tures: analysis of experimental dose-response curves and otoxic and teratogenic effects of semicarbazide. Toxicology statistical comparison with theoretical effects. Arch. Com- 36, 183–198. plex Environ. Stud. 9, 23–33. Selye, H., 1957. . Rev. Can. Biol. 16, 1–82. Po¨ch, G., Dittrich, P., Holzmann, S., 1990. Evaluation of Smith-Mungo, L.I., Kagan, H.M., 1998. Lysyl oxidase: prop- combined effects in dose-response studies by statistical erties, regulation, and multiple functions in biology. Ma- comparison with additive and independent interactions. J. trix Biol. 16, 387–398. Pharmacol. Meth. 24, 311–325. Steel, R.G.D., Torrie, J.H., 1980. Principles and Practices of Po¨ch, G., Dawson, D.A., Reiffenstein, R.J., 1996. Model Statistics: A Biometrical Approach, second ed. McGraw- usage in the evaluation of combined effects of toxicants. Hill, New York 633 pp. Toxicol. Ecotox. News 3, 51–59. Tang, S.S., Trackman, P.C., Kagan, H.M., 1983. Reaction of Riggin, G.W., Schultz, T.W., 1986. Teratogenic effects of aortic lysyl oxidase with b-aminopropionitrile. J. Biol. benzoyl hydrazine on frog embryos. Trans. Am. Microsc. Chem. 258, 4331–4338. Soc. 105, 197–210. Wang, S.X., Mure, M., Medzihradszky, K.F., Burlingame, Schultz, T.W., Ranney, T.S., 1988. Structure-activity relation- A.L., Brown, D., Dooley, D.M., Smith, A.J., Kagan, ships for osteolathyrism. II. Effects of four-position alkyl H.M., Klinman, J.P., 1996. A crosslinked cofactor in LO: substitution of formic acid hydrazide. Toxicology 53, 147– function for amino side chains. Science 273, 1078– 159. 1084.

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