Although the fold increase varied by stage (GZ, 166-fold; PrM, 216-fold; M, 545-fold; PoM, 171-fold), the stage-related pattern (immature > mature) and the magnitude of the gradient (3- to 4-fold) remained the same (compare Figs. 3 and 5). In comparison to levels in testis, basal Cd levels in nongonadal tissues were much higher, but the increase after CdCl2 treatment was of lower magnitude (29-, 72-, and 105-fold in liver, heart, and kidney, respectively). ampicillin antibiotic
Tissue 109Cd-Binding Activity (Fig. 6)
A standard MT assay was used to measure basal and Cd-induced changes in heat-stable Cd-binding activity. Binding activity was stage dependent (PrM = PoM > GZ = M) in untreated animals, but zonal differences did not correspond to those predicted by tracer or atomic absorption spectrophotometry analysis. Three days after a single injection of radioinert CdCl2, MT-like activity increased in all but M stages (Fig. 6), and the response in GZ (4-fold) was of greater magnitude than in PrM and PoM stages (2.5-and 1.8-fold; Fig. 6). In untreated animals, MT-like binding activity in kidney and liver was 3 to 15 times higher than in testis, but CdCl2 induced similar fold increases in all three tissues (3- to 4-fold). Interestingly, induction of the MT-like component was greatest in the heart (23-fold). Comparison of Figures 5 and 6 shows that the MT-like protein accounted for only a small fraction of total bound Cd, regardless of tissue type or treatment.
Subcellular Distribution of 109Cd (Fig. 7)
After injection of tracer, radioactivity (cpm/g) in cytosolic, nuclear, and membrane subfractions reflected levels in whole homogenates and whole tissues (compare Figs. 3 and 7). Regardless of tissue type or stage, proportionally more 109Cd was recovered from cytosol (~75%) than from other subfractions; however, fractional recovery of radioactivity from cell nuclei in GZ/PrM stages (14% and 13%, experiments 1 and 2, respectively) was > 2-fold greater than radioactivity in this subfraction in other stages or in kidney (< 6%, both experiments).
FIG. 6. Differences in 109Cd-binding activity and effect of Cd pretreatment in A) staged testicular tissues and B) control tissues. Animals were untreated (n = 8) or were given a single injection of CdCl2 3 days before being killed (n = 3). Dissected tissues were processed to obtain extracts, and 109Cd-binding activity was analyzed in the heat-resistant fraction (see Materials and Methods). Values were expressed in ^g 109Cd bound/g original tissue wet weight and represent the mean of all animals per treatment group with duplicate determinations per animal. A) Data were analyzed by two-way ANOVA to determine significance by stage (p < 0.001) and treatment (p < 0.001). Regional differences in a given treatment group were determined by a post hoc comparison test (p < 0.05), and those with different letters differed significantly: untreated, a, b; Cd treated, k, l. For a given region, differences between control and treated groups are indicated by an asterisk. In B, different tissue types were not compared statistically. For a given tissue, differences between control and treated groups were determined by Student’s t-test (*, p < 0.05).
FIG. 7. Stage-related differences in the subcellular distribution of 109Cd. Animals received a single injection of 109Cd and were killed after 3 days (n = 1, experiment 1) or 7 days (n = 2, experiment 2). Dissected tissues were processed to salt-resistant and salt-extractable nuclear fractions, cytosol, and membranes (plus mitochondria). Radioactivity was measured in triplicate in each fraction. Values for each fraction were expressed as cpm/g tissue.