Human hepatocellular carcinomas (HCC) from patients in Qidong, an area of high incidence in China, in which both hepatitis B virus and aflatoxin B1 are risk factors, were analysed for mutations in p53, a putative tumour-suppressor gene. Eight of the 16 HCC had a point mutation at the third base position of codon 249. The G----T transversion in seven HCC DNA samples and the G----C transversion in the other HCC are consistent with mutations caused by aflatoxin B1 in mutagenesis experiments. No mutations were found in exons 5,6,8 or the remainder of exon 7. These results contrast with p53 mutations previously reported in carcinomas and sarcomas of human lung, colon, oesophagus and breast; these are primarily scattered over four of the five evolutionarily conserved domains, which include codon 249 (refs 4-9). We suggest that the mutant p53 protein may be responsible for a selective clonal expansion of hepatocytes during carcinogenesis.

A G:C-->T:A mutational hotspot at codon 249 of the p53 tumor suppressor gene has previously been identified in hepatocellular carcinoma (HCC) of patients from Qidong, China and southern Africa in which aflatoxin B1 (AFB1) and hepatitis B virus (HBV) are known synergistic risk factors. We have examined p53 mutation patterns of HCC from geographic areas in which the risk factors vary. Nine HCC lines and four hepatoblastoma lines (HB) were examined for p53 gene mutations and the relationship with HBV infection. Five of the nine HCC lines had homozygous mutation or deletion randomly distributed in exons 6-8, whereas none of the four HB cell lines had p53 mutations. One of the four HB lines (HepG2) had an N-ras mutation at codon 61 position 2. The p53 point mutations in the three HCC cell lines from Japan resulted in the amino acid changes of cysteine for tyrosine in cell line HuH 7 at codon 220 (A:T-->G:C), alanine for glycine in cell line HLF at codon 244 (G:C-->C:G), and serine for arginine in cell line HLE at codon 249 (G:C-->C:G). In addition, the deletion of 18 base pairs from codon 264 position 3 to codon 270 position 1 has resulted in the deletion of Leu-Gly-Arg-Asn-Ser-Phe from the amino acids sequences 256-270 in the Japanese cell line HuH 4. The cell line PLC/PRF/5 that showed p53 mutation at codon 249 (G:C-->T:A) with substitution of serine for arginine was derived from a South African patient. Our results indicate that whereas the p53 gene is not mutated in the HB cell lines, the HCC cell lines frequently contain an abnormal p53 gene. In addition, p53 point mutations were not detected in the four Japanese HCC cell lines that were positive for genomic integration of HBV X-gene and surface antigen gene. The three Japanese HCC cell lines with p53 mutations did not contain HBV sequences, indicating that hepatocarcinogenesis associated with p53 mutation does not require the genomic integration of HBV sequences.

Neutral red (NR) in medium was absorbed and concentrated in lysosomes of cultured rat and human hepatocytes. NR uptake increased with the time of incubation and reached a plateau in 2 hr. Uptake was proportional to the concentration of the NR solution and the numbers of viable liver cells. Prolonged culture of hepatocytes increased the numbers of lysosomes, and thus, the dye accumulation. The NR can be extracted from lysosomes for quantitative measurement of hepatocyte viability and cytotoxicity of xenobiotics. With this assay, several serum-free media (e.g., Waymouth's, MEM, LHC-8, etc.) were compared for the maintenance of viable hepatocytes in vitro. Interestingly, LHC-8 medium, which is used to grow human bronchial epithelial cells, best preserved viable rat hepatocytes. The cytotoxic effects of dimethylnitrosamine (DMN) and aflatoxin B1 (AFB1) were examined by NR assay on rat and human hepatocyte cultures and were found to be dependent on dose and time of the exposures. NR50 was 20 mM for DMN and 0.072 microM for AFB1 in rat hepatocytes with 24 hr of exposures and reduced to 12.5 mM for DMN and 0.053 mu microM for AFB1 with 48 hr exposures. Human hepatocytes were more resistant to the toxicity of both chemicals; NR50 values were 100 mM DMN and 1.8 microM AFB1 respectively, for 24 hr treatments. Compared with lactate dehydrogenase (LDH) leakage test, the NR assay was simpler and more sensitive in determining the viability and cytotoxicity of xenobiotics in primary cultures of hepatocytes.

Rotavirus and enterotoxin-producing bacteria are major causes of diarrheal disease in humans. A method of rapid diagnosis, ultrasensitive enzymatic radioimmunoassay, has been developed to quantitatively detect cholera toxin and rotavirus. The method uses features of both enzyme-linked immunosorbent assay and radioimmunoassay; however, the sensitivity of the assay is 100-to 1000-fold more sensitive than the two parent assays. Ultrasensitive enzymatic radioimmunoassay should also be useful in measuring other biologically important agents such as drugs and hormones.In recent years, the capability to detect small quantities of biologically important molecules has been greatly expanded by the development of radioimmunoassays (RIA) (1, 2). These assays have been especially useful for the detection of circulating substances, such as hormones, drugs, and infectious agents. However, RIA have certain practical limitations. The short half-life of the isotopes used limits the shelf life of the reagents. In addition, the need for y-emitting isotopes subjects the users of RIA to a radiation hazard. Enzyme immunoassays (also known as enzyme-linked immunosorbent assays or ELISA) have been developed in an attempt to overcome those problems (3-5). ELISA is similar in design to solid-phase RIA except that an enzyme is used as the immunoglobulin marker instead of a 'y-emitting isotope. This enzyme-antibody conjugate is bound to the solid phase by a series of antibody-antigen reactions and essentially converts the substrate to products with a visible yellow color, which can be measured spectrophotometrically. The fact that a single molecule of enzyme is capable of reacting with a large number of substrate molecules provides for amplification and, thus, a high degree of sensitivity (3, 6). Because stable enzymes such as alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] can be used, ELISA also has the advantage of using reagents with a long shelf life.In practice, however, the sensitivity of ELISA systems has not significantly exceeded that of RIA (7). In order to combine the advantages of both RIA and ELISA, we developed a practical ultrasensitive enzymatic radioimmunoassay (USERIA) for the detection of antigens such as human rotavinus (an important cause of infantile gastroenteritis) and cholera toxin. These assay systems are a 1000-fold more sensitive than both RIA and ELISA for the detection of these agents. MATERIALS AND METHODSReagents.[3H]AMP (generally 3H-labeled, 15 Ci/mmol, New England Nuclear; 1 Ci = 3.7 X 10O°becquerels) was purified by column chromatography with DEAE-Sephadex (A-25; Pharmacia) in a stepwise manner as follows: (i)