Ruth E. Gibson-D'Ambrosio

Antibodies to UV irradiated DNA: the monitoring of DNA damage by Elisa and indirect immunofluorescence

Abstract The enzyme‐linked immunosorbant assay (ELISA) was modified to (1) characterize antibodies raised in rabbits against UV‐irradiated single‐stranded DNA (UVssDNA) complexed with methylated BSA and (2) directly detect pyrimidine dimers in irradiated DNA. The antisera specifically bound to UVssDNA, UVpoly(dT) and to a limited extent to UVdsDNA and UVpoly(dC) immobilized on protamine sulfate coated microliter wells. Fifty percent of the maximum antibody binding was observed at a 1‐5000 dilution against UVssDNA. Binding to ssDNA and poly(dT) was observed only at much higher concentrations of antibody (1:500 dilution), whereas no binding to double stranded DNA (dsDNA) was observed. The extent of binding of the antibody was dependent on the dose of UV radiation to DNA, as well as, to the concentration of antigen immobilized on the plate. Specific binding to DNA irradiated with 5.0 J/m2 was detected with as little as 10 ng of DNA. The sensitivity was further extended to less than 1 J/m2 by using higher concentrations (100 ng) of UVssDNA. The ability of various irradiated molecules, DNA, homopolymers and linkers to act as inhibitors of antibody binding establish that the antigenic determinants are mainly thymine homodimers with lower affinity for cytosine dimers. Potential usefulness of the antibodies to directly quantitate pyrimidine dimers in cells exposed to UV radiation was determined by indirect immunofluorescence. Flow cytometric analysis of immunostained human lymphocytes irradiated with 254 nm radiation indicated that greater than 50% of the population had significantly higher fluorescent intensity than unirradiated control cells.

Mechanisms of nitric oxide–induced cytotoxicity in normal human hepatocytes

Chronic exposure of hepatocytes to reactive nitrogen species (RNS) following liver injury and inflammation leads not only to functional and morphological alterations in the liver but also to degenerative liver diseases and hepatocellular carcinoma. Previously, we showed that S‐nitroso‐N‐acetylpenicillamine‐amine (SNAP), which generates nitric oxide, and 3‐morpholinosydnonimine (Sin‐1), which generates equal molar concentrations of superoxide and nitric oxide resulting in peroxynitrite production, exhibited different levels of cytotoxicity to normal human hepatocytes in culture. The aim of the present study was to elucidate some of the molecular and cellular pathways leading to hepatocyte cell death induced by RNS. Following treatment of the hepatocytes with SNAP or Sin‐1, gene‐specific DNA damage was measured in mtDNA and a hprt gene fragment using a quantitative Southern blot analysis. Both agents induced dose‐dependent increases in DNA damage that was alkaline labile, but not sensitive to both formamidopyrimidine‐DNA glycosylase (fpg) and endonuclease III, which recognize 8‐oxoguanine, thymine glycol, and other oxidized pyrimidines. DNA damage was two‐ to fivefold greater in mtDNA than in the hprt gene fragment. There was a persistent and marked increase in DNA damage posttreatment that appeared to arise from the disruption of electron transport in the mitochondria, generating reactive species that saturated the repair system. DNA damage induced by Sin‐1 and SNAP led to cell‐cycle arrest in the S‐phase, growth inhibition, and apoptosis. The data support the hypothesis that the functional and morphological changes observed in liver following chronic exposure to RNS are, in part, the result of persistent mitochondrial and nuclear DNA damage. Environ. Mol. Mutagen. 37:46–54, 2001

The establishment and continuous subculturing of normal human adult hepatocytes: Expression of differentiated liver functions

The use of normal adult liver hepatocytes in cell culture for biochemical, toxicological and pharmacological studies has been greatly limited owing to the loss of replicative capacity and differentiated liver function. This is contrary to the ability of the liver to regenerate following injury in vivo. This suggests that liver “stem” or “transitional” hepatocytes exist that upon proper stimulus divide and differentiate into mature hepatocytes. In this study we report the establishment and culture of hepatocytes from normal human adult liver, which: (1) possess replicative capacity sufficient to subpassage 12–15 times (27–37 cumulative population doublings); (2) can be cryopreserved for subsequent use without loss of replica five capacity; and (3) upon differentiation in culture synthesize albumin and keratin 18 and metabolize benzo[a]pyrene. The ability of these cells to divide or express differentia tedfunctions appears to be due to a number of cellular, biochemical and physical characteristics that are present during the primary establishment and subsequent growth phases of the cell cultures. Disassociation of cells ffom excess liver tissue was best achieved by combining the mechanical action of the Stomacher@ with very low amounts of proteolytic enzymes and EGTA. The cell lines appeared to grow best when established and subpassaged in an rnALPHA medium supplemented with insulin, hydrocortisone, transferrin, epithelial growth factor and fetal bovine serum ® rescreened for human hepatocyte cell growth). The seeding density and cell-cell contact in culture appeared to be important for both cell division and expression of liver function. When cells were seeded at a low density and subpassaged before confluency, the cells continued to divide. Albumin and keratin 18 synthesis occurred primarily in tightly packed cell clusters. When cells were seeded at a high density, near confluency, albumin and keratin 18 synthesis occurred uniformly in all of the cells of the culture and the culture metabolized benzo[a]pyrene to water-soluble metabolites, which covalently bound to cellular DNA. This appearance of liver functions was consistent with the “transition” of hepatocytes to a terminally differentiated state. Nonhepatic markers, i.e., α-fetoprotein, factor VIII and ψ-glutamyl transpeptidase activity were not expressed in cells cultured at either low or high density. Thus, the data presented here indicate that normal human adult liver hepatocytes, once established in culture, can be subpassaged to a high number of population doublings, cryopreserved for later use, and modulated to express differentiated liver functions.bl]References

The regenerative capacity of adult human hepatocytes: clonal expansion of hepatocytes producing albumin in culture

Abstract Upon injury, the liver has the capability to replace lost hepatocytes by at least two processes. In the unipotential model hepatocytes exit quiescence after injury and replicate, while in the multipotential model non-parenchymal epithelial cells replicate and differentiate into mature hepatocytes. To test the former process, we cloned normal adult human hepatocytes expressing differentiated function, and monitored the continued expression of these differentiated functions as they proliferated and expanded in cell culture. Fifty-eight cell clones, derived from two different normal adult human liver primary cell lines, expressing albumin and cytokeratin 18, but not cytokeratin 19, as markers of hepatocyte differentiation, were isolated. These cell clones were serially subpassaged, five to eight times, and the expression of hepatocyte markers was determined using immunocytochemistry. Of the 58 clones isolated, 56 (96%) were positive for both albumin and cytokeratin 18, and two were negative for albumin but positive for cytokeratin 18. Upon subpassage of the clones, the cells continued to expand before senescence at a cumulative population doubling of 27.1±0.8. All of the cell strains, originating from clones producing albumin and cytokeratin 18, continued to express these two markers through senescence. The data indicate that mature normal human hepatocytes are capable of producing progeny hepatocytes, supporting the unipotential model for replacement of hepatocytes in the human liver.