Margaret M. Manson

Production of Polyclonal Antisera

All immunochemical procedures require a suitable antiserum or monoclonal antibody raised against the antigen of interest Polyclonal antibodies are raised by injecting an immunogen into an animal and, after an appropriate time, collecting the blood fraction containing the antibodies of interest. In producing antibodies, several parameters must be considered with respect to the final use to which the antibody will be put. These include (1) the specificity of the antibody, i.e., the ability to distinguish between different antigens, (2) the avidity of the antibody, i.e., the strength of binding, and (3) the titer of the antibody, which determines the optimal dilution of the antibody in the assay system. A highly specific antibody with high avidity may be suitable for immunohistochemistry, where it is essential that the antibody remains attached during the extensive washing procedures, but may be less useful for immunoaffinity chromatography, as it may prove impossible to elute the antigen from the column without extensive denaturation.All immunochemical procedures require a suitable antiserum or monoclonal antibody raised against the antigen of interest Polyclonal antibodies are raised by injecting an immunogen into an animal and, after an appropriate time, collecting the blood fraction containing the antibodies of interest. In producing antibodies, several parameters must be considered with respect to the final use to which the antibody will be put. These include (1) the specificity of the antibody, i.e., the ability to distinguish between different antigens, (2) the avidity of the antibody, i.e., the strength of binding, and (3) the titer of the antibody, which determines the optimal dilution of the antibody in the assay system. A highly specific antibody with high avidity may be suitable for immunohistochemistry, where it is essential that the antibody remains attached during the extensive washing procedures, but may be less useful for immunoaffinity chromatography, as it may prove impossible to elute the antigen from the column without extensive denaturation.

Effect of hexachlorobenzene on male and female rat hepatic gamma-glutamyl transpeptidase levels

The effect of 200 ppm hexachlorobenzene (HCB) in the diet on hepatic gamma glutamyl transpeptidase (GGT) levels was examined in F344 male and female rats. During early stages of feeding (4-29 weeks) some females had elevated GGT levels spread throughout the periportal regions, but all those examined at 29 weeks had a few GGT positive foci. The livers of males were not affected at this time. However, after 90 weeks of feeding, males also had elevated periportal GGT activity and a number of GGT positive foci. All females had lost the periportal activity but had developed GGT positive hepatocellular tumours. It therefore appeared that elevated nonfocal GGT levels are a poor indicator for HCB toxicity, but GGT positive foci may be a more reliable indicator of carcinogenic changes.

Immunohistochemical detection of BromodeoxyurIDine-Labeled Nuclei for In Vivo Cell Kinetic Studies

The classical technique for identifying cells engaged in DNA synthesis is by their uptake of [(3)H]-thymidine, detected using autoradiography. However, this method can be inconvenient, as specialized darkroom and radioisotope facilities are required, with the potential health hazard that handling isotopes entails. Bromodeoxyuridine (BrdU), the halogenated 5-substituted derivative of deoxyuridine, is a thymidine analog specifically incorporated into the DNA of proliferating cells during S phase. This is now a well-established alternative to (3)H thymidine, since it has been shown that labeling indices for the two molecules are the same (1,2). The development of a monoclonal antibody (3) that recognizes BrdU incorporated into single-stranded DNA has resulted in several techniques using immunocytochemical staining to detect incorporated BrdU in frozen, paraffin- and plastic-embedded sections of tissue by light microscopy. It has also proved extremely valuable for studies in conjunction with flow cytometry and even, for in vivo studies of human tumor cell kinetics (see this vol., Chapter 43 ). We describe here a method to detect DNA synthesis by in vivo labeling of nuclei with BrdU, followed by indirect immunological detection in paraffin-embedded tissue (4).The classical technique for identifying cells engaged in DNA synthesis is by their uptake of [(3)H]-thymidine, detected using autoradiography. However, this method can be inconvenient, as specialized darkroom and radioisotope facilities are required, with the potential health hazard that handling isotopes entails. Bromodeoxyuridine (BrdU), the halogenated 5-substituted derivative of deoxyuridine, is a thymidine analog specifically incorporated into the DNA of proliferating cells during S phase. This is now a well-established alternative to (3)H thymidine, since it has been shown that labeling indices for the two molecules are the same (1,2). The development of a monoclonal antibody (3) that recognizes BrdU incorporated into single-stranded DNA has resulted in several techniques using immunocytochemical staining to detect incorporated BrdU in frozen, paraffin- and plastic-embedded sections of tissue by light microscopy. It has also proved extremely valuable for studies in conjunction with flow cytometry and even, for in vivo studies of human tumor cell kinetics (see this vol., Chapter 43 ). We describe here a method to detect DNA synthesis by in vivo labeling of nuclei with BrdU, followed by indirect immunological detection in paraffin-embedded tissue (4).

double Label Immunohistochemistry on Tissue Sections Using Alkaline Phosphatase and PeroxIDase Conjugates

One of the most common methods in immunohistochemistry involves the use of an antibody to the antigen of interest detected indirectly with an enzyme-labeled antispecies secondary antibody. The enzyme catalyzes the formation of a colored insoluble reaction product at the antigen site. It is possible, with careful choice of reagents, to label two antigens simultaneously, resulting in two different colored reaction products (1). Cells or tissue sections can also be double-labeled with two antispecies secondary antibodies carrying different fluorochromes (see this vol., Chapter 42 ), or by using suitable antibodies conjugated to different sizes of colloidal gold (see this vol., Chapter 19 ).One of the most common methods in immunohistochemistry involves the use of an antibody to the antigen of interest detected indirectly with an enzyme-labeled antispecies secondary antibody. The enzyme catalyzes the formation of a colored insoluble reaction product at the antigen site. It is possible, with careful choice of reagents, to label two antigens simultaneously, resulting in two different colored reaction products (1). Cells or tissue sections can also be double-labeled with two antispecies secondary antibodies carrying different fluorochromes (see this vol., Chapter 42 ), or by using suitable antibodies conjugated to different sizes of colloidal gold (see this vol., Chapter 19 ).

The production of carcinomas in vivo by the intravenous or subcutaneous injection of a hepatocellular tumour cell line

Suspensions of cells of an aflatoxin-induced hepatocellular carcinoma cell line, which grow rapidly as tumours when injected subcutaneously into nude mice, have been injected subcutaneously and also intravenously via the hepatic portal or tail vein into syngeneic host animals. Efficiency of the injections was checked histologically and histochemically in parallel experimental animals. Subcutaneous injections consistently resulted in the appearance of solid tumours at the site of injection within 1 month. However, despite the apparent importance of the establishment of a blood supply when tumours are grown subcutaneously, only a low incidence of tumours (less than 20%) was detected in animals receiving intravenous injections. These results suggest that the proposed use of injection of liver cell suspensions into the portal tract as a sensitive means of detecting transformation may only be of limited value.

Sex Difference in the Susceptibility of Rats to the Development of Porphyria and Hepatic Tumours caused by Hexachlorobenzene

The hypolipidaemic agent nafenopin (Su-13437) belongs to a class of structurally diverse compounds which induce a proliferation of the hepatic peroxisome compartment after short-term treatment. Life-long feeding of such compounds is associated with an increased incidence of liver tumours. However, the mechanism by which such compounds induce these tumours is not known, nor is it clear whether a causal relation exists between the peroxisome proliferation and the hepatocarcinogenicity. Nafenopin is not mutagenic in the standard Ames test or in several other tests for genotoxic activity. However, nafenopin is known to induce certain mono-oxygenase activities and to alter the pattern of cytochrome P-450 isoenzymes. Consequently, it may be more appropriate to assess the mutagenic activity of nafenopin by using an activating system isolated from animals pretreated with the compound. Nafenopin also induces several peroxisomal oxidase activities, particularly fatty acid ~-oxidation. Thus it is conceivable that excess hydrogen peroxide production associated with this enzyme induction may lead to oxidative DNA damage as suggested by Reddy et al. (1980). We have tested both these possibilities. Nafenopin was not mutagenic to Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538 or TA 102 either directly or after activation by S-9 fractions isolated from control, Aroclor 1254or nafenopin-treated rats, in the presence or absence of uridine diphosphate (UDP)-glucuronic acid. However, treatment of rats with nafenopin (200 mg/kg for 7 days) or the structurally related clofibrate (300 mg/kg for 7 days) was accompanied by slight increases in the amount of hepatic nuclear DNA eluted from polycarbonate filters (3 )JLm pore size) under alkaline conditions. In a 5 h elution period (6 ml/h) 6.7% of the DNA from control rats was eluted from the filters (n = 23). This value was increased about 3.5-fold by clofibrate treatment (n = 8) and 2-fold by nafenopin treatment (n = 11). Further studies suggested that hydrogen peroxide was not responsible for the observed effects. The significance of these findings is under investigation.