Hazard evaluation of chemicals that cause accumulation of alpha 2u-globulin, hyaline droplet nephropathy, and tubule neoplasia in the kidneys of male rats.

This review paper examines the relationship between chemicals inducing excessive accumulation of alpha 2u-globulin (alpha 2u-g) (CIGA) in hyaline droplets in male rat kidneys and the subsequent development of nephrotoxicity and renal tubule neoplasia in the male rat. This dose-responsive hyaline droplet accumulation distinguishes CIGA carcinogens from classical renal carcinogens. CIGA carcinogens also do not appear to react with DNA and are generally negative in short-term tests for genotoxicity, CIGA or their metabolites bind specifically, but reversibly, to male rat alpha 2u-g. The resulting complex appears to be more resistant to hydrolytic degradation in the proximal tubule than native, unbound alpha 2u-g. Single cell necrosis of the tubule epithelium, with associated granular cast formation and papillary mineralization, is followed by sustained regenerative tubule cell proliferation, foci of tubule hyperplasia in the convoluted proximal tubules, and renal tubule tumors. Although structurally similar proteins have been detected in other species, including humans, renal lesions characteristic of alpha 2u-g nephropathy have not been observed. Epidemiologic investigation has not specifically examined the CIGA hypothesis for humans. Based on cancer bioassays, hormone manipulation studies, investigations in an alpha 2u-g-deficient strain of rat, and other laboratory data, an increased proliferative response caused by chemically induced cytotoxicity appears to play a role in the development of renal tubule tumors in male rats. Thus, it is reasonable to suggest that the renal effects induced in male rats by chemicals causing alpha 2u-g accumulation are unlikely to occur in humans.

Long-term course of surgically induced astigmatism.

We performed an analysis of surgically induced astigmatism in 229 cases of extracapsular cataract extraction and posterior chamber lens implantation. The average length of follow-up for patients in this study was 34.4 months (2.87 years). We found that surgically induced astigmatism continued to change for at least three years after surgery. The preoperative astigmatism was found to have only minimal effect on the postoperative astigmatism if the corneal curvature was controlled with keratometry at the time of surgery. The optimal amount of with-the-rule astigmatism at three to five weeks postoperatively was found to be 0.75 diopter to 1.25 diopters for one surgeon and surgical technique.

Differential effects of transforming growth factor-beta on proliferation of normal and malignant rat liver epithelial cells in culture.

Transforming growth factors (TGF-betas) have been shown to cause both stimulatory and inhibitory effects on cellular growth in a variety of normal and neoplastic cells. The nature of the inhibitory effects of TGF-beta on proliferation of different cell types is at present unclear. We have used freshly isolated rat hepatocytes, a normal diploid rat liver epithelial cell line (NRLM), and a subline (AFB) derived from it which was transformed in vitro by aflatoxin B1 to study the nature of TGF-beta-induced growth inhibition and its alteration following chemically induced neoplastic transformation. TGF-beta had a vastly different effect on proliferation of normal rat liver epithelial cells (both freshly isolated and NRLM cells) compared to aflatoxin B1-transformed cells. TGF-beta at 20 pg/ml caused 83% inhibition of colony formation of NRLM, whereas the growth of AFB cells was unaffected by TGF-beta at concentrations as high as 10 ng/ml. A parallel dose-dependent inhibition of DNA synthesis by TGF-beta was observed in both primary hepatocytes and NRLM cells at concentrations between 10 pg and 10 ng/ml. No inhibition of DNA synthesis was observed in AFB cells. Furthermore, TGF-beta did neither induce anchorage-independent growth of NRLM cells nor affect the growth of AFB cells in soft agar. TGF-beta-induced inhibition of the NRLM cells was irreversible in nature, since treated cells were unable to proliferate and form colonies upon removal of TGF-beta from the medium. Also, NRLM cells showed, after 4 days in the presence of 20 pg of TGF-beta per ml morphological changes characterized by cytoplasmic hypertrophy and the formation of abundant liposomal derivatives, some of which resemble lipofuscin. The finding that TGF-beta caused a high degree of irreversible inhibition of NRLM cells emphasizes the need for caution in interpreting data from inhibition studies, since most assays presently used are designed for assessing growth stimulation in vitro and do not adequately distinguish between the possible cytotoxic and/or cytostatic action of growth inhibitors.

The effects of nonfixated lower lens haptics of Binkhorst lenses on corneal endothelial cell density.

At specified intervals up to 56 months postoperatively, we took central corneal endothelial cell measurements of 52 patients who had undergone extracapsular cataract extraction (ECCE) with placement of a Heyer-Schulte IC-10 iridocapsular intraocular lens. We then compared the endothelial cell density of patients in whom the lower lens haptic was fixated within the capsule with those in whom it was nonfixated. We found that nonfixation of the lower lens haptic, which in essence converts the irido-capsular lens to an iris-supported lens, does not cause continuing endothelial cell loss.

Differences between men and women as related to intraocular lens implantation.

We examined 291 cases of IOL implantation in order to evaluate differences between males and females for several factors. We found that women have shorter axial lengths, older age at time of surgery, greater preoperative corneal curvature, higher power of IOL implanted, more surgically induced astigmatism, and a higher rate of complications. Insignificant differences between the two groups included pre- and postoperative refraction, preoperative astigmatism, visual acuity, type of IOL implanted, corneal thickness, endothelial cell count, and percent of patients requiring discissions.

Measurement of cell proliferation in microculture using Hoechst 33342 for the rapid semiautomated microfluorimetric determination of chromatin DNA.

We report the development and characterization of a semiautomated method for measurement of cell proliferation in microculture using Hoechst 33342, a non-toxic specific vital stain for DNA. In this assay, fluorescence resulting from interaction of cell chromatin DNA with Hoechst 33342 dye was measured by an instrument that automatically reads the fluorescence of each well of a 96-well microtiter plate within 1 min. Each cell line examined was shown to require different Hoechst 33342 concentrations and time of incubation with the dye to attain optimum fluorescence in the assay. In all cell lines, cell chromatin-enhanced Hoechst 33342 fluorescence was shown to be a linear function of the number of cells or cell nuclei per well when optimum assay conditions were employed. Because of this linear relation, equivalent cell doubling times were calculated from growth curves based on changes in cell counts or changes in Hoechst/DNA fluorescence and the fluorimetric assay was shown to be useful for the direct assay of the influence of growth factors on cell proliferation. The fluorimetric assay also provided a means for normalizing the incorporation of tritiated thymidine ( [3H] TdR) into DNA; normalized values of DPM per fluorescence unit closely paralleled values of percent 3H-labelled nuclei when DNA synthesis was studied as a function of the concentration of rat serum in the medium. In summary, the chromatin-enhanced Hoechst 33342 fluorimetric assay provides a rapid, simple, and reproducible means for estimating cell proliferation by direct measurement of changes in cell fluorescence or by measurement of changes in the normalized incorporation of thymidine into DNA.

Factors associated with poor predictability by intraocular lens calculation formulas.

In this study, we evaluated and compared two groups of posterior chamber intraocular lens (IOL) implantation cases. One group consisted of patients whose postoperative refraction was accurately predicted by IOL calculation formulas, while the other group included patients whose postoperative refraction was poorly predicted by the same formulas. We found that although postoperative astigmatism was greater in the poorly predicted group, preoperative to postoperative changes in astigmatism did not differ between the two groups. The poorly predicted group also had a shorter average axial length, a greater proportion of females, and an increased variability in most of the measurements we performed. The two groups did not differ significantly in terms of measured postoperative anterior chamber depth, age at the time of the surgery, IOL power and style implanted, complication rate, or preoperative corneal integrity.

Clinical evaluation of six intraocular lens calculation formulas.

We examined the results of posterior chamber intraocular lens (IOL) implantation and evaluated six commonly used IOL power calculation formulas (original Binkhorst, modified Binkhorst, Colenbrander, Shammas, Hoffer, and SRK regression) to determine which ones produce the most accurate and predictable results. We found that the accuracy of the various formulas depends upon several factors, including the surgeon's technique, the type and style of posterior chamber IOL implanted, and the axial length of the eye being operated on. Furthermore, we found that the results using the different formulas vary in a consistent pattern. Surgeons must therefore evaluate their cases periodically to determine which formulas and IOL styles will provide their patients with the most accurate and satisfying results.

Rapid determination of DNA synthesis in adherent cells grown in microtiter plates.

A specific, rapid, and economical method for measuring the extent of DNA synthesis in adherent rat hepatoma H4-II-E cells grown in 96-well microtiter plates is described. The adherent cells were pulsed for 1 h with [methyl-3H]thymidine, released from the substratum by trypsinization, and collected on fiberglass filters with a MASH II cell harvester. The amount of radioactivity incorporated was directly proportional to the number of cells per well. Growth curves generated by measuring [methyl-3H]thymidine incorporation and counting the number of cells per well were identical. Experiments with inhibitors of DNA, protein, and RNA synthesis demonstrated that this method selectively measured DNA synthesis. In addition, [3H]thymidine uptake showed excellent correlation with autoradiographic assessment of DNA synthesis. This specific and sensitive method for determining DNA synthesis in microtiter cultures should facilitate studies of effects of various growth-controlling agents on epithelial, fibroblastic, and other cells which grow as adherent cells in culture.

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.

Expression of gamma-glutamyl transpeptidase activity in the developing mouse tooth, intervertebral disc, and hair follicle.

Expression of gamma-glutamyl transpeptidase (GGT) in the developing mouse tooth, intervertebral disc, and hair follicle was investigated in terms of its localization during ontogenic stages and its association or lack of association with cell proliferation (labeled nuclei after [3H]thymidine injection or metaphase-arrested cells after colchicine injection). The data demonstrate that (a) GGT expression followed a program of activity and localization changes that correlated with the progressive emergence of developmental stages and (b) GGT activity in developing tissues derived either from epithelium (enamel-producing cells and hair follicle cells) or from mesenchyme (intervertebral disc cells) was localized only in mitotically quiescent cellular layers or regions associated with the production of specialized tissue products; however, not all postmitotic regions expressed GGT activity. Although further research is needed to clarify the role of GGT in normal and neoplastic tissues, we conclude that increasing evidence from this and other laboratories implicates GGT as a marker of cell differentiation, cell aging, and/or reduced cell proliferation.

Changes in liver lobule glycogen zonation during prolonged fasting of rats previously fed a 30% casein diet and adapted to a controlled feeding schedule.

Changes in the lobular distribution of liver glycogen were studied during the prolonged fasting of young adult male Sprague-Dawley rats that were previously adapted to a 30% casein diet and to the 2 + 22 controlled feeding and lighting schedule. The optical density of glycogen, revealed in fixed liver cryostat sections by the periodic acid-Schiff procedure, was determined in the periportal (P), midlobular (M), and centrilobular (C) regions of the liver lobule at various times during a 196-hour fasting period. The prolonged fast could be divided into 3 phases with respect to liver glycogen variation: initial glycogen depletion, glycogen resurgence, and final glycogen depletion. Glycogen was lost from all regions of the liver lobule during the initial glycogen depletion phase. During the glycogen resurgence phase, a lobular glycogen concentration gradient formed (P greater than M greater than C). The simultaneous occurrence of increasing liver glycogen, increasing liver tyrosine aminotransferase activity, and decreasing body fat during glycogen resurgence suggests that the young adult rat does not spare body protein during starvation. During the final glycogen depletion phase, liver glycogen was again present in a lobular gradient (P greater than M greater than C) but the absolute amount of glycogen in each region of the lobule was markedly reduced in comparison with rats killed during glycogen resurgence.

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.

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.

Scanning microdensitometry of glycogen zonation in the livers of rats adapted to a controlled feeding schedule and to 30, 60, or 90% casein diets.

The effect of diet composition on diurnal changes in glycogen zonation patterns in rat liver was investigated in individually-caged male Sprague-Dawley rats adapted to the 2 + 22 controlled feeding and lighting schedule and to diets containing 30% casein/55% carbohydrates, 60% casein/25% carbohydrates, or 9.0% casein (30 rats/dietary group). Three rats from each dietary group were killed at the following times relative to the onset of feeding (0 min):--60, --30, 0, 15, 30, 45, 60, 90, 120, and 180 min. Glycogen in cryostat sections from the median and right lateral lobes of the liver was fixed and stained by standard techniques. The optical density of glycogen at points along the path between the central and portal veins of a given lobule was determined, and lobular glycogen gradients of replicate animals were integrated to form a composite lobular glycogen distribution profile. In the period from--60 to 0 min, liver glycogen levels were similar for rats on any of the diets, and the glycogen concentration was similar in periportal (P), midlobular (M), and centrilobular (C) hepatocytes. During the 0- to 45-min period, diet-related glycogen depletion occurred (90 > 60 > 30% casein) by asymmetrical glycogen loss (P > M > C hepatocytes) from the liver lobules. Similar food intake curves occurred for all diets. During the 45- to 180-min period, asymmetrical glycogen accumulation began in lobular parenchymal cells (P > M > C hepatocytes), and rate of accumulation was related to dietary to dietary composition (30 > 60 > 90% casein). The differential responses of parenchymal cells within liver lobules to physiological stimuli resulted in glycogen distribution changes that were rapid and of large magnitude. Our results are consistent with the hypothesis that periportal and midlobular hepatocytes are more metabolically responsive and active than centrilobular hepatocytes

Metabolite control of L-fucose utilization.

Thyroid fucokinase is responsive to a number of metabolites which might serve in a regulatory capacity. In addition to inhibition by ADP and stimulation by GMP, fucokinase responds selectively to a series of nucleotide sugars. Of those studied, only guanine nucleotide sugars moderate the activity of the enzyme. GDP-alpha-D-mannose, GDP-alpha-D-glucose, GDP-alpha-D-rhamnose, and GDP-alpha-L-fucose on the other hand is strongly inhibitory. In the case of GDP-alpha-D-mannose stimulation, a physiological role seems possible, but the rationale is not entirely clear. The effects of GDP-beta-L-fucose, on the other hand may represent physiological control effected through feedback inhibition by and end product.