Target tissue morphology and serum biochemistry following 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in a TCDD-susceptible and a TCDD-resistant rat strain.

The mode of action of the highly toxic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is unknown. It was recently discovered that two strains of rat, Long-Evans (L-E) and Han/Wistar (H/W), differ widely in susceptibility to TCDD. Employing this strain divergence as a probe, the present study set out to assess the role of various biochemical and morphological effects in TCDD lethality. In the main experiment, the rats were treated once ip with 0,5,50, or (H/W) 500 micrograms/kg TCDD and killed 1 to 16 days postexposure. Several target organs were evaluated by light microscopy and a number of serum lipid and carbohydrate parameters as well as a few major regulatory hormones were analyzed. The results demonstrated that most alterations caused by TCDD were essentially similar in both strains. TCDD reduced circulating thyroxine to a slightly greater extent and more permanently in the sensitive L-E strain. Moreover, a highly significant interaction on thyroid-stimulating hormone was found among strain, dose, and time. Serum concentrations of corticosterone and free fatty acids were increased only in the L-E rats given 50 micrograms/kg TCDD, i.e., at an apparent LD100 dose level for this strain. Yet, the most striking interstrain difference was seen in the liver which was distinctly affected after Day 4 in L-E rats given 50 micrograms/kg TCDD but only marginally affected in rats from any H/W group. The lesion, while showing no necrotic cell changes, was suggestive of plasma membrane damage, possibly reflecting the production of free radicals. The relation of the findings to possible mechanisms of TCDD action is discussed.

Quantitative immunohistochemistry of metallothionein in rat placenta.

The presence of metallothionein (MT) was demonstrated in placentae from cadmium-exposed and control rats by an immunohistochemical technique, using peroxidase as label and the diaminobenzidine procedure for the staining reaction. The protein was found in different regions of the placenta, i.e. in trophoblastic labyrinth, in spongiotrophoblast and in visceral yolk sac. Cytophotometric analysis of the final reaction product revealed that the amount of MT was increased in the placental labyrinth of cadmium-exposed rats. Increases were found in both nuclei and cytoplasm of trophoblast cells in the labyrinth region. Possible roles of MT in the transport of zinc and in the carbohydrate metabolism are discussed.

Differences in immunological susceptibility to cadmium toxicity between two rat strains as demonstrated with cell biological methods. Effect of cadmium on DNA synthesis of thymus lymphocytes.

When 2 inbred rat strains, the Brown-Norway rat and the Lewis rat were exposed to the same amount of CdCl2 for 15 days, a completely different immunological reaction pattern could be demonstrated. Despite the same amount of intrathymic cadmium in both strains, the Brown-Norway rat showed a significant decrease in thymocytes in the S-phase and a significant increase of thymocytes in the G2 phase and mitosis, in contrast with findings in the Lewis rats. A new method for estimating subtle forms of thymus atrophy showed a slight decrease in the number of the smallest thymocytes in the Brown-Norway rat after exposure to cadmium, in contrast with that in the Lewis rat. Evidence is presented that the approximately 1.7 times larger number of thymocytes/mg thymus in the Lewis rat, compared to the Brown-Norway rat, as well as the approximately 2.5 times lower proliferation rate of the thymocytes, and an approximately 1.5 times higher metallothionein content of the thymus medulla epithelial cells in the Lewis rat, might be responsible for the observed difference in toxicity. The zinc content of the thymus was not significantly decreased by exposure to CdCl2, and did not differ significantly between both strains.

Increase of cadmium-thiolate clusters as a measure of morphological non-toxic cadmium accumulation in the rat liver.

When cadmium is chronically administered to rats, an increase by more than 10% of protein bound disulphides and cadmium-thiolate clusters appears to be an indicator for non-toxic accumulation of cadmium in liver and kidney and probably in other organs as well. Using enzyme histochemistry, no damage could be observed in these livers, on the contrary, even signs of increased cellular activity could be demonstrated with specific staining for single stranded RNA. It is clearly demonstrated that in the case of 2 livers with the same quantity of accumulated cadmium morphological damage is completely dependent on dose and schedule of administration. However, despite the fact that cadmium is retained very well in rat livers showing an increase in protein-bound disulphides and cadmium-thiolate clusters, there are still small morphological changes, especially in cells and tissues that appear to have a relatively small potency for producing cadmium-binding proteins.

A new combined quantitative cytophotometric method for estimating sulphydryl groups or disulphide bonds, and total protein in liver sections; applied to rats chronically exposed to cadmium.

A detailed procedure is described for a new combined quantitative cytophotometric method for estimating sulphydryl groups or (and) disulphide bonds as well as total protein. When applied to livers of cadmium treated rats and appropriate controls a dose response effect was demonstrated for the quantity disulphide bonds per unit protein.

Histochemical staining of cadmium thiolate clusters in livers of rats treated chronically with cadmium.

In livers of rats exposed to varying doses of CdCl2 80-90% of the cadmium content present in the fresh tissue is retained if these livers are fixed with a neutral or acid formalin fixative. Cadmium assays during different stages of the staining procedure for protein bound disulphides show the ability of this staining to demonstrate cadmium thiolate clusters next to disulphides. The methods described may also be useful in gaining more insight in the mechanism involved in fixation and staining procedure of some other metals.

Large increase in disulphide bonds containing cytosol proteins after chronic cadmium administration, estimated in isolated rat liver cells.

Using histochemical staining, followed by cytophotometric quantitation of disulphide bonds and total protein in isolated liver cells of rats treated for a long time with low doses of CdCl2, a large increase in disulphide bonds containing proteins could be demonstrated in cells of one ploidy class. This increase seems to be due to an increase in high molecular weight (HMW) cytosol proteins as estimated biochemically. They probably represent polymers of metallothionein.

Histochemical changes in protein disulphide bonds in rat liver and kidney after chronic cadmium administration, and the possible relation to metallothionein.

After chronic exposure to low doses of CdCl2 an increase in disulphide bonds has been established in rat liver using a specific staining method for disulphide bonds and cytophotometric quantitation. This increase is dependent on doses and length of exposure time. Evidence is presented that this increase might be related to the accumulation of metallothionein or some other cadmium binding protein. Using the same staining method after long exposure to low doses of CdCl2 a large number of large dark blue stained granules were observed in the proximal tubule cells, with blue stained deposits in the lumen of the proximal and some renal medulla tubules of the kidney. Evidence is presented that this staining pattern corresponds to the destruction of the proximal tubule cell by the cadmium thionein complex.

Investigation of the mechanism of cadmium toxicity at cellular level. I. A light microscopical study.

Chromatin condensation occurs within 15 min to 1 h after the addition of CdCl2 in a concentration of 1 microgram/ml to a liver cell culture and seems to be the first event demonstrable by light microscopy. This chromatin condensation, which precedes membrane leakage, is irreversible and leads to cell death of almost all cells. It does not occur after the administration of equimolecular concentrations of some other bivalent metallic ions.

Investigation for the mechanism of cadmium toxicity at cellular level. II. An electron microscopical study.

With quantitative techniques at electron microscopical level chromatin condensation and emptying of the interchromatin space have been established in the nuclei of the endothelial cells of small uterine vessels. The nuclear and cytoplasmic changes after cadmium administration show much similarity between endothelial cells of small uterine vessels and cultured liver parenchymal cells. Cytoplasmic changes in both cell types after cadmium administration are suggestive of a disturbance in ribosomal RNA synthesis as the main cause leading to ultimate cell lysis.

Cytophotometric, flow cytofluorometric and morphometric demonstration of the existence of an antigen dependent thymus lymphocyte subpopulation.

Using absorption cytophotometry and flow cytofluorometrical DNA and protein estimation of single thymus lymphocytes we were able to establish that after injection of a large dose of antigen (ovalbumin) a subpopulation of lymphocytes arises in the thymus with high protein contents above that of those lymphocytes normally present, however, in small quantities in the thymus. By morphometrical analysis it was established that these lymphocytes are situated in the outermost cortex.

A cytophotometric and flow cytofluorometric approach to the differentiation of T lymphocytes in the thymus.

By cytophotometric and flow cytofluorometric DNA and protein determinations two main proliferating subpopulations of thymus lymphocytes with a different percentage of cells in the S phase could be distinguished. One subpopulation had a very low protein content, was cortisone sensitive and located in the cortex. Cells with comparable low protein contents were not found amongst lymphocytes of the peripheral blood. The other lymphocyte subpopulation had a higher protein content, was cortisone resistant and situated in the cortex around a group of epithelial cells and in the medulla. The protein content of these thymus lymphocytes appeared to be comparable to that of the peripheral blood lymphocytes. On the basis of the protein content per cell, it is possible to identify and isolate the more often described major subpopulation of cortisone sensitive thymus lymphocytes remaining and dying in the thymus, and the minor cortisone resistant subpopulation of thymus lymphocytes which is the source of the peripheral T lymphocyte.

Quantitative histological aspects of liver cirrhosis.

In human liver cirrhosis and other liver diseases relatively large nuclei are found with finely meshed chromatin and large nucleoli which do not occur in liver of individuals without liver disease. The number of tetraploid nuclei near central veins and portal areas in normal livers is not statistically significantly increased in comparison with the surrounding areas. The percentage of diploid nuclei in these livers varies from 80-95 per cent. There are definite differences in the number of adjacent microscopical fields of view with an increased degree of polyploidy when livers of normal individuals are compared with livers from patients with suspected liver disease and patients with liver cirrhosis. The increased degree of polyploidy in the last two groups can be considered as sign of regeneration.

Microphotometry of rat liver nucleoproteins during the cell cycle, and comparison of diploid nuclei in the G2 period with tetraploid nuclei.

The following facts were established with a microphotometric investigation of isolated nuclei from rat liver in different stages of the cell cycle. During the mitotic wave occurring in the liver of newborn animals after injection of casein it was found that the naphtol yellow S (NYS) protein content of the nuclei increases about 30% during the G1-period. A second increase of around 70% was established during the S-phase whereas no increase could be observed during the G2-phase. An indication for the existence of a "critical protein mass" of the nuclei before the onset of the S-phase could be observed. The protein content of diploid nuclei in the G1-phase of adult animals is about 50% higher than in newborns. This makes it probable that there is no significant difference in the NYS-protein content of diploid nuclei in the G2-period and tetraploid nuclei of adult rats. No differences were observed between diploid nuclei in the G2-period of newborn rats and tetraploid nuclei of adult rats in their Fastgreen histon, RNA and SH plus SS content. The only criterion to distinguish between G2 nuclei and tetraploid nuclei seems to be the number of nucleoli, but this is rather unreliable.