Global bioethics: converting sustainable development to global survival.

Millions of people in various parts of the world and within each country are presently surviving in categories described as "mere," "miserable," "idealistic," "irresponsible," and "acceptable." The term "acceptable survival" is proposed as a bioethical goal of global survival, looking beyond the 21st century to the year 3000 and beyond. The frequently used alternative term is "sustainable development," but in most contexts this is an economic concept and does not imply any moral or ethical constraints, except where these are spelled out. Acceptable survival, broadly defined, means acceptable to a universal sense of what is morally right and good and what will continue in the long term. The expanding dominant, but irresponsible, world culture is not an acceptable type of development because it cannot survive in the long term.

Cultured hepatoma cells for the study of enzyme regulation: induction of ornithine decarboxylase by insulin and asparagine.

The induction and decay of ornithine decarboxylase (ODC) by insulin and asparagine in cultures of H4-II-EC3 (H35) hepatoma cells was studied in a modified Waymouth medium in the presence of fetal bovine serum (FBS) and in serum-free media. The insulin response was enhanced by the presence of asparagine although the effect of asparagine was not so much on the initial increase as it was on a slowing of the decline after the maximum was reached at 6 to 8 h after the supplements were added together with fresh medium. In all cases the initial ODC activity was zero at zero time for addition of media and supplements, and, after reaching the maximum, activity declined to near zero by 24 h. Fetal bovine serum gave induction that followed a similar time course but was inferior to the combination of insulin plus asparagine and, in fact, FBS inhibited the latter response. Putrescine (the product formed from ornithine by ODC), at 10(-5) M, markedly inhibited the induction of ODC by insulin or FBS, but the inhibition was less when asparagine was present.

Alternative hypotheses for the role of promotion in chemical carcinogenesis.

A new protocol for carcinogenesis in rat liver is described in order that confirmatory experiments might be undertaken concurrently. The basic protocol, designated IPI (initiator + promoter + initiator), is presented in several alternative forms, including the possible use of X-irradiation as the initiator. The rationale is discussed in terms of the two-hit somatic mutation theory of Armitage and Doll, with an initial hit produced by the first dose of initiator and expansion of single cells to sizable clones by promotion thereby increasing the probability of a second hit by the second dose of initiator. The question of relevant mutations was taken up and it was proposed that genes for chalones (C) and for chalone receptors (R) are logical targets for consideration in a two-mutation sequence. Alternative hypotheses pertaining to promoter action were described in terms of possible mechanisms by which nonelectrophilic promoters might simulate a second mutation by increasing or decreasing the levels of a nonchromosomal replicating particle in target cells.

gamma-Glutamyl transpeptidase and alpha-fetoprotein expression during alpha-naphthylisothiocyanate-induced hepatotoxicity in rats.

Continuous feeding of alpha-naphthylisothiocyanate to young male Sprague-Dawley rats was shown to produce a concentration-dependent increase in the number of hepatic ductular cells and a concentration- and time-dependent elevation of serum and liver gamma-glutamyl transpeptidase and alpha-fetoprotein. In liver, the increased gamma-glutamyltranspeptidase and alpha-fetoprotein were predominantly confined to the proliferative ductular cell population. It is concluded that early stages of intoxication by the noncarcinogen alpha-naphthylisothiocyanate resemble early stages in induction of liver neoplasia by carcinogens that evoke ductular proliferation. Elevation of gamma-glutamyltranspeptidase and alpha-fetoprotein expression by an expanding ductular cell population characterizes both processes. However, the increase is rapidly reversed after alpha-naphthylisothiocyanate is discontinued, in contrast to the persistence that has been reported when acetylaminofluorene was administered.

Effects of hormones and amino acid depletion on the kinetic parameters of amino acid uptake in monolayer cultures of rat hepatocytes.

Uptake of 2-aminoisobutyric acid (AIB) at concentrations of 0.1 mM to 30 mM was examined in sodium-containing and sodium-free media in hepatocytes pretreated without hormones (control), with hormones, or with amino acid depletion. Results show that 1-minute but not 4-minute rates can be taken as initial rates for the total or sodium-dependent transport of AIB. The data for the 1-minute sodium-dependent transport of AIB were analyzed by a computer program and also by Eadie-Hofstee and Lineweaver-Burk plots, and a single saturable system was found. In the control cultures, the saturable system had a Km of 1-2 mM AIB and a Vmax of 1.2 nmoles AIB/mg protein/minute. There was an increase in the Vmax of two to three-fold after pretreating the cultures with insulin or amino acid depletion, three to four-fold with glucagon, and six to seven fold with glucagon + dexamethasone.

Phenotypic diversity of gamma-glutamyltranspeptidase activity and protein secretion in hepatoma cell lines.

Characteristic and patterns of gamma-glutamyltranspeptidase (GGT) expression were studied in four rat and three human hepatoma cell lines. Phenotypic diversity of GGT expression was demonstrated by the following findings. (a) GGT specific activity increased rapidly in three of four rat lines during the first 72 hr after subculture. (b) GGT activity was detected in the fourth rat cell line only from 96 to 120 hr after subculture. (c) In late log or stationary cultures, each of the four rat lines assumed a unique and characteristic level of GGT specific activity. (d) The intracellular GGT distribution pattern was markedly varied in rat and human cell lines. (e) GGT activity was confined to isolated cell clusters in one human line in vitro and one rat line both in vivo and in vitro. And (f) there was poor correlation between GGT specific activity and several liver-associated and hepatoma-associated properties. In contrast to evidence of diversity in GGT expression, GGT was shown to be a nonsecreted protein in all four rat cell lines. The constitutive or autogenous nature of the GGT phenotype in rat hepatoma cells was demonstrated by the retention of the GGT-positive and GGT-negative phenotypes of two strains grown in mixed culture; the lack of change in GGT activity when cells were cultured on different substrata, in different media, or in media containing hormones (insulin, dexamethasone, triiodothyronine, or glucagon); and the assumption of nearly constant levels of GGT specific activity in late log or stationary cultures. The results suggest that GGT activity is expressed in hepatomas as a result of disturbed differentiation and that this expression is not necessarily linked to cell proliferation.

Application of quantitative stereology to the evaluation of enzyme-altered foci in rat liver.

The mathematical science of quantitative stereology has established relationships for the quantitation of elements in three-dimensional space from observations on two-dimensional planes. This report describes the utilization and importance of such mathematical relationships for the quantitative analysis of focal hepatic lesions in terms relative to the volume of the liver. Three examples are utilized to demonstrate the utility of such calculations in the three-dimensional quantitation of hepatic focal lesions. The first is that of a computer-simulated experiment based on defined hypothetical situations. The simulations demonstrate the applicability of the computations described in this report to the evaluation of two-dimensional data from typical animal experiments. The other two examples are taken from actual experiments and involve the transplantation of hepatic cell populations into the liver suitably prepared hosts and the quantitation of altered foci produced by initiation with diethylnitrosamine-partial hepatectomy followed by promotion with phenobarbital. The quantitation of altered foci by means of a two-dimensional analysis (simple enumeration of focal intersections/area of tissue section) is proportional to the quantitation of foci per volume of liver provided that the mean diameter of the foci for each treatment is sufficiently uniform, as exemplified in the text by the transplantation experiment. When such mean diameters are unequal as in the diethylnitrosamine-phenobarbital experiment described herein, quantitation from three-dimensional analysis gives significantly different results as compared with enumeration of focal intersections on two-dimensional areas. These studies clearly demonstrate that the frequency and size of foci intersections viewed on two-dimensional tissue sections do not necessarily reflect the number of size of foci in the three-dimensional tissue. Only by quantitating the number and size of the foci in relation to the three-dimensional volume of the tissue can one determine the validity of the proportionality of data from two-dimensional measurements to the total number of foci per volume of tissue. Such a conclusion has important implications for quantitative studies on hepatocarcinogenesis as well as for the enumeration of premalignant lesions which occur during the natural history of carcinogenesis in any solid tissue.

Hepatic cyst formation in male rats partially hepatectomized as weanlings during short-term feeding of 2-acetylaminofluorene.

Partial hepatectomy (p.h.) of weanling male Sprague-Dawley rats during short-term feeding of 0.03% 2-acetylaminofluorene (AAF) resulted, 12 to 15 months later, in a massive production of hepatic cysts. The weight of the right lateral and caudate lobes plus neoplasms (the left lateral and median lobes were excised at the time of p.h.) accounted for as much as 43% of total body weight. The incidence of hepatic cysts was negligible when animals were not subjected to p.h. during AAF feeding. The feeding of 0.05% phenobarbital (PB) subsequent to AAF + p.h. did not significantly increase the incidence of hepatic cysts. By comparison of the present data with that of other investigators, it can be suggested that susceptibility to cholangioma formation following AAF + p.h. may be determined in part by the developmental stage of the rat and/or its liver at the time of the AAF + p.h. regimen.

A new protocol and its rationale for the study of initiation and promotion of carcinogenesis in rat liver.

A new protocol for carcinogenesis in rat liver is described in order that confirmatory experiments might be undertaken concurrently. The basic protocol, designated IPI (initiator + promoter + initiator), is presented in several alternative forms including the possible use of X-irradiation as the initiator. The rationale is discussed in terms of the two-hit somatic mutation theory of Armitage and Doll, with an initial hit produced by the first dose of initiator and expansion of single cells to sizable clones by promotion thereby increasing the probability of a second hit by the second dose of initiator. The question of relevant mutations is taken up and it is proposed that genes for chalones and for chalone receptors are logical targets for consideration in a two-mutation sequence.