A.J. Gandolfi

Dynamic organ culture of precision liver slices for in vitro toxicology

The lack of a reproducible method for the production of thin tissue slices has hindered the use of liver slices as an in vitro tool for hepatotoxicity studies. Fresh human, rat, and rabbit liver was processed using a mechanical slicer. With this instrument, precision (5% of thickness) liver slices in the submillimeter range could be produced at a rapid rate. Slices were prepared from fresh livers in chilled, oxygenated buffer to minimize trauma. Following incubation for up to 20 h in a dynamic organ culture system, histology of incubated slices suggested that 250 m precision-cut slices were optimum in regard to morphology relative to liver slices incubated under conventional organ culture conditions. Addition of bromobenzene to the culture showed time-dependent hepatotoxicity based on two classic parameters of cell degeneration. Histological evidence is presented which suggests the usefulness of this system for hepatotoxicity studies and the production of focal necrosis in vitro.

Maintenance of Adult-rat Liver Slices in Dynamic Organ-culture

SummaryAdult rat liver slices were maintained for 20 h in a novel organ culture system with minimal loss of viability and function. Potassium and adenosine triphosphate levels were maintained at in vivo levels, following an initial recovery period (2 to 4 h), for up to 20 h. Protein synthesis and secretion were linear for 20 and 16 h, respectively. In addition, the liver slices synthesized glycogen between 4 and 12 h in culture. Finally, the liver slices were hormonally responsive during the 20 h culture period as exemplified by glucagon-stimulated glucose production. This system provides a simple and effective method for the culture and biochemical maintenance of adult rat liver for 20 h with minimal loss of biochemical function.

Cold- and cryopreservation of human liver and kidney slices

Tissue slices may provide a rapid and economical way of determining cold ischemic effects on human liver and kidney cell viability and metabolism. In contrast to isolated hepatocyte cultures, tissue slices offer an in vitro system which more closely resembles the in vivo situation because of the differentiation and functional heterogeneity of the slice. In this study, human liver and kidney slices were cold stored for 10 days in Belzers University of Wisconsin (UW), Euro-Collins, and Modified Sacks solutions. Another set of slices was cryopreserved at 1 degree C/min for liver and 12 degrees C/min for kidney using a 10% dimethyl sulfoxide/fetal calf serum (FCS) cryoprotectant solution. The cold- and cryopreserved slices were incubated in roller culture for 4 h using FCS as the media. Liver slice viability was assessed by K+ content, protein synthesis, gluconeogenesis, and urea synthesis. Kidney slice viability was assessed using K+ content, protein synthesis, and organic ion transport (PAH and TEA). Human kidney slices were cold preserved in UW for 4-6 days, while the human liver slices were preserved for 12-24 h depending on the viability parameter. Following cryopreservation, human liver slice viability was retained at between 65 and 90% of control values, while kidney slice viability was maintained between 70 and 90% of control values depending on the viability parameter. These results indicate that this human in vitro tissue slice system can be used to optimize preservation solutions and methods. The ability to cold- and cryopreserve human slices could facilitate the more efficient utilization of human tissue.

Biotransformation activity in vitrified human liver slices

In vitro testing of human liver for biotransformation or xenobiotic metabolism studies has been limited by unpredictable acquisition of samples. Consequently, it has become necessary to consider methods to cryopreserve and store these samples whenever they do become available for culture of the revived tissue at a more convenient time. Human liver slices were cryopreserved by vitrification, which allows for the transfer of aqueous media to low temperatures (-196 degrees C) without the formation of ice crystals. Human liver slices were exposed to increasing concentrations of 1,2-propanediol up to a final concentration of 4.76 M in fetal calf serum. Slices were then vitrified by direct immersion into liquid nitrogen and warmed by submersion in 37 degrees C fetal calf serum. Warming was done either immediately or after 4 and 8 weeks of storage under liquid nitrogen. The effects of vitrification, storage time, and warming on biotransformation were determined by assessing the integrated metabolism of 7-ethoxycoumarin (7-EC). Vitrified or fresh human liver slices were exposed to 50 microM 7-EC and its primary metabolite 7-hydroxycoumarin (7-HC) in organ culture for up to 6 hr. Metabolite production of both fresh and vitrified liver slices was compared. Retention of the inherent biotransformation rate was usually high and seemed independent of storage time. Integration of both cytochrome P450-mediated and secondary conjugation processes was retained in cryopreserved tissue. Vitrification offers a way to cryopreserve human liver slices for the study of xenobiotic metabolism in humans.

Human liver quality is a dominant factor in the outcome of in vitro studies

Donated human liver in the form of precision-cut tissue slices or isolated hepatocytes, is increasingly being used to predict metabolism and toxicity of xenobiotics in man. These tissue slices or hepatocytes can also be cold-preserved and cryopreserved to prolong their use for biological experiments. The viability of human liver could substantially affect the outcome of such experimentation. The goal of this investigation was to assess the viability of donated human livers, in the form of tissue slices, as they were received and to determine how varying degrees of liver quality affect experimental outcomes. Over one hundred human livers were categorized according to initial viability, as assessed by ATP content, K+ retention, protein synthesis, and LDH leakage. Each liver was placed in a low-, a medium-, or a high-quality group. The results showed that 76% of transplant-grade tissue (procured for transplantation) fell into the high-viability classification while the majority of research-grade tissue (not procured for transplantation) fell into the lowest viability classification. It was also found that only tissue slices prepared from highly viable human liver could be cold-preserved and cryopreserved. Dichlorobenzene metabolism was also greater in slices from highly viable human livers as compared to less viable livers. This study showed that human liver tissue acquired for medical research substantially varies in its viability and that these differences will affect the experimental data obtained.

Toxicity and metabolism of subcytotoxic inorganic arsenic in human renal proximal tubule epithelial cells (HK-2)

Arsenic is an environmental toxicant and a human carcinogen. The kidney, a known target organ of arsenic toxicity, is critical for bothin vivo arsenic biotransformation and elimination. This study investigates the potential of an immortalized human proximal tubular epithelial cell line, HK-2, to serve as a representative model for low level exposures of the human kidney to arsenic. Subcytotoxic concentrations of arsenite (≤10 μmol/L) and arsenate (<100 μmol/L) were determined by leakage of LDH from cells exposed for 24 h. Threshold concentrations of arsenite (between 1 and 10 μmol/L) and arsenate (between 10 and 25 μmol/L) were found to affect MTT processing by mitochondria. Biotransformation of subcytotoxic arsenite or arsenate was determined using HPLC-ICP-MS to detect metabolites in cell culture media and cell lysates. Following 24 h, analysis of media revealed that arsenite was minimally oxidized to arsenate and arsenate was reduced to arsenite. Only arsenite was detected in cell lysates. Pentavalent methylated arsenicals were not detected in media or lysates following exposure to either inorganic arsenical. The activities of key arsenic biotransformation enzymes – MMAV reductase and AsIII methyltransferase – were evaluated to determine whether HK-2 cells could reduce and methylate arsenicals. When compared to the activities of these enzymes in other animal tissues, the specific activities of HK-2 cells were indicative of a robust capacity to metabolize arsenic. It appears this human renal cell line is capable of biotransforming inorganic arsenic compounds, primarily reducing arsenate to arsenite. In addition, even at low concentrations, the mitochondria are a primary target for toxicity.

Comparative toxicity of valproic acid and its metabolites in liver slices from adult rats, weanling rats and humans

The incidence of fatal hepatic failure associated with valproic acid (VPA) treatment is low but when it occurs, 90% of the affected patients are below the age of 20 yr. At present the mechanism of VPA hepatotoxicity is unclear; however, it may be a combination of the formation and action of toxic metabolites in developing tissues. In the study reported here we investigated the action of VPA and its metabolites in liver slices prepared from adult and weanling Sprague-Dawley rats and from human livers (non-transplantable livers from organ donors, or biopsy material from patients undergoing surgical liver resection). VPA, 2-propyl-2-pentenoic acid (2-en-VPA or Delta(2)-VPA), 2-propyl-4-pentenoic acid (4-en-VPA or Delta(4)-VPA) were incubated for various times at concentrations of 100 or 300 mug/ml. Protein synthesis and K(|) content were used to assess functional integrity or general viability. The question addressed was whether there were differences in the in vitro toxicity of VPA and its metabolites that were related to the age of the livers from which the slices were taken. Liver slices from weanling rats were significantly more sensitive to VPA and its metabolites than the slices from livers of adult rats. The rank order of toxicities (4-en-VPA > VPA > 2-en-VPA) was the same in both sets of rat slices. The human liver slices were significantly affected by VPA and its metabolites but these compounds were equal in their ability to produce this toxicity. There was also an indication of differences in sensitivity to the toxicity of VPA in slices from livers of donors of different ages.

Ennancement of DMN-induced Kidnex Tumors by 1,2–Dichlorovinylcysteine in Swiss-Webster Mice

ABSTRACT1,2–Dichlorovinylcysteine (DCVC) is known to cause enlarged nuclei in renal proximal tubule epithelium. This study further characterized the cellular changes induced by DCVC. Also a preliminary investigation of the initiator and promoter potential of DCVC was conducted. When given for 4 weeks in drinking water to S-wiss-Webster mice, DCVC (50 or 100 μg/ml) caused a progressive change in renal epithelia which was persistent at 23 weeks. Enlarged nuclei with uneven chromatin dispersal, multiple nucleoli, and irregular nuclear membranes resided in enlarged, abnormally-shaped cells. These changes were resolved by week 50. Mice initiated with dimethylnitrosamine developed renal tumors by week 27. Mice fed DCVC (10 or 50,μg/ml) for 14 weeks following dimethylnitrosamine initiation, had a slightly higher tumor incidence, a higher incidence of invasive tumors, and had multiple renal tumors unlike animals given dimethylnitrosamine alone.

Toxic responses to defined chemical mixtures: mathematical models and experimental designs.

The problem and relevance of assessing biological responses to chemical mixtures is presented with reference to the literature on this problem and its possible solutions. This review is intended for a general audience as an introduction to, and comment on, assessing the interactions of defined mixtures of xenobiotics. The focus is on experimental toxicology, however, the methods are also applicable to pharmacology. Much of the literature on this topic is quite specialized in statistics, theory, or specific applications. This may deter a significant portion of the growing number of investigators in this field from using this literature, and may partially account for the persistent use of methods which have been shown to permit precarious conclusions. References are given for some of the most comprehensive and recent work and reviews on the subject. The reader is given some familiarity with this topics basic problems and ideas, and the controversy on terminology. One example is presented of a popular experimental design and data analysis method which while applicable in some situations, has been shown to lead to precarious and even erroneous conclusions. Eight other methods of data analysis are briefly presented and some of their advantages, disadvantages, assumptions, and limitations are discussed. These methods were selected to illustrate similarities and differences in the various approaches taken in addressing this problem. Three basic types of experimental design appropriate to these kinds of studies are outlined. General considerations, suggested guidelines, and possible pitfalls in experimental design, and data analysis of biological responses to chemical mixtures are discussed.

Organ culture of rat myocardial slices: An alternative in vitro tool in organ-specific toxicology

SummaryPrecision-cut adult rat myocardial slices of highly consistent dimensions were produced and maintained for at least 24 h in an organ culture system. These relatively thin (350–400 pm) slices were generated from tissue cores (7 mm diameter) obtained from coring the sagittally slabbed myocardium. The tissue cores were then used to produce slices with a mechanical tissue slicer under specific temperature (25°C) and buffer (zero Ca2+, high K+) conditions. Myocardial slices are viable for at least 24 h in culture, as assessed by a biochemical battery including K+ and Ca2+ content, labeled leucine incorporation, ATP content, and the loss of the cytoplasmic enzyme LDH. In addition to these parameters, histological evaluation was also employed. These data indicate that the precision-cut myocardial slice system is a possible alternative in vitro tool and merits further study in the field of cardiotoxicity.