hibitory effect. In long-term exposure to sublethal 
concentrations of heavy metals, therefore, an animal’s 
biochemistry may be adapted in ways that will mask 
effects observed in acute-static studies. 
In the initial seven experiments of this study, 
results of which are published throughout this 
collaborative report, skeletal muscle was the only 
tissue available in sufficient quantity for biochemical 
testing. Although the relatively slow metabolism of 
the muscle would not be expected to reflect the 
biochemical response of rapidly metabolizing liver, for 
instance, the muscle sarcoplasm was, nevertheless, 
examined for possible changes in malic enzyme (ME)? 
and a-glycerophosphate dehydrogenase activities, 
which in vertebrates require manganese and 
magnesium, respectively, for optimal activity. 
Subsequent experiments provided liver and blood, 
where early reaction to physiological challenge would 
logically be expected. In one experiment, the test con- 
centrations of cadmium were 0, 3, and 24 ppm, with 
nine fish at each concentration; the livers were tested 
for aspartate aminotransferase (AAT) activity, a trans- 
aminase that reflects metabolic stress in vertebrates 
(e.g., Amador and Wacker, 1965) and invertebrates 
(e.g., Hammen, 1969) alike. In another experiment, 
only two concentrations of cadmium were used (0 and 
24 ppm), with 14 fish at each concentration; the livers 
of these fish were tested for changes in ligand response 
of NAD reductase activity. The sera were subjected to 
electrophoresis and examined for possible changes in 
total-protein patterns that might reflect an increasing 
metal-protein fraction, for carbonic anhydrase activi- 
ty (a zinc-enzyme), and for esterase activity. 
METHODS AND MATERIALS 
Cunners were exposed to 0, 3, and 24 ppm cadmium 
for 96 hr in experiments subsequent to those described 
by Calabrese, Collier, and Miller (this report, Part I), 
under the same test conditions. The blood was drawn 
as described by Thurberg and Dawson (this report, 
Part III); the livers were excised, pooled, and placed 
in small plastic pouches from which as much air as 
possible was excluded before freezing. In several of the 
initial experiments that included more concentrations 
of cadmium (Calabrese et al., this report, Part I), the 
fillets were cut from the cunner frames, skinned, and 
packaged and frozen in the same way as the livers. 
Treatment of Tissue 
Liver.—The freshly excised livers from three fish 
were pooled for each sample and were frozen-stored 
* Abbreviations used in this report are: ME=malic enzyme, 
E.C.1.1.1.40; @GPdH=alpha-glycerophosphate dehydrogenase, 
E.C.1.1.1.8: NAD=nicotinamide-adenine dinucleotide, and NADH= 
the reduced form; NADR-Mg=magnesium-dependent NAD re- 
ductase activity: and ATT = asparate aminotransferase, E.C.2.6.1.1. 
until use, for no longer than 1 wk. The pooled livers 
were homogenized with a glass pestle in chilled, 
double-distilled water, 1:9 (w/v) for the AAT assay 
and 1:4 or 1:9 for the NADR-Mg assay. Homogenates 
were centrifuged for 45 min at 14,500 g and 4°C, and 
the supernates used as the crude enzyme preparations 
(E). For AAT assays of the freshly frozen livers, it was 
necessary further to dilute the supernates 1:9 with 
iced water, for a final E dilution factor of 100. 
Skeletal muscle.—Paired fillets, frozen-stored 
from 4 to 6 wk, were pounded to a rough paste with an 
iced mortar and pestle and centrifuged for 45 min at 
14,500 g and 4°C. No suspending medium was used. 
The centrifuged tissue fluid (CTF) served as the en- 
zyme preparation for ME and a@GPdH assays. 
Blood.—The clotted fresh blood was clarified by 
centrifugation for 30 min at 1,720 g and 4° C, and the 
resulting serum was used for electrophoresis. 
Assay Procedures 
The water used in preparing all solutions was 
doubly glass-distilled; solutions of substrate and 
coenzyme were prepared fresh daily; and the assays 
were read on a double-beam, ratio-recording spec- 
trophotometer, in an optical cuvette with a 1-cm path 
length. Change in absorbance at 340 nm from 30 to 
90 sec after the beginning of the reaction was taken 
as the unit of measurement (A A*° X 10°/min/0.10 ml 
EB). 
Aspartate aminotransferase.—The procedure 
used was essentially that of Bergmeyer and Bernt 
(1963), except for the proportions used of reagent 
solutions. No malic dehydrogenase was added. H, the 
supernate from the liver homogenate, had a dilution 
factor of 100. 
Protocol: 
Buffer-substrate solution: = 2.70 ml 
Phosphate buffer (0.1 M, pH 7.6), 
and K aspartate (0.25 M) 
NADH solution (10 mg/ml H,O) = 0.10 ml 
E preparation = 0.10 ml 
The solutions were pipetted into an optical cuvette 
and allowed to stand for 10 min at room temperature. 
Absorbance was read against a reference cuvette (con- 
taining medium with no initial NADH); the reference 
mixture was adjusted with small increments of 
NADH so that the difference in absorbance between 
sample and reference was no greater than 0.600. The 
reaction was not started until there was no detectable 
oxidation of NADH. Substrate (0.10 ml 0.2 M 
potassium a-ketoglutarate) was added to start the 
reaction. 
