Effect of Iron Deposition on Native T1 Mapping and Blood Oxygen Level Dependent for the Assessment of Liver Fibrosis in Rabbits With Carbon Tetrachloride Intoxication.

RATIONALE AND OBJECTIVES: This study aimed to explore the effect of iron deposition on native T1 mapping and blood oxygen level-dependent (BOLD) imaging in detecting liver fibrosis (LF) in a rabbit model. MATERIALS AND METHODS: An LF group (n = 100) was established by subcutaneously injecting 50% carbon tetrachloride (CCl4) oil solution, and 20 normal rabbits composed a control group. Native T1 mapping and BOLD were performed, and the T1native and R2* quantitative parameters were analyzed by receiver operating characteristic (ROC) and multiple logistic regression analyses, with histopathological results and liver iron content (LIC) serving as reference standards. RESULTS: In total, 18, 17, 16, 18, and 15 rabbits were histopathologically diagnosed with LF stages F0, F1, F2, F3, and F4, respectively. T1native (r = 0.47), R2* (r = 0.75) and LIC (r = 0.61) increased with LF stage progression (p < 0.001). Compared to T1native values, R2* performed better in diagnosing the LF stage, especially for distinguishing F1-F2 from F3-F4 (AUC = 0.66 vs. 0.91, p = 0.01). Combined with the LIC, both T1native and R2* showed improved diagnostic value in comparison to the individual imaging techniques, particularly for diagnosing F0 vs. F1-F2 and F0 vs. F1-F4, with AUC values of 0.90 vs. 0.70 (p = 0.01) and 0.93 vs. 0.77 (p = 0.01) for T1native + LIC vs. LIC, respectively. CONCLUSION: BOLD imaging performed better than native T1 mapping in predicting and diagnosing LF stage progression. The decrease in diagnostic accuracy caused by the deposition of liver iron is a potential pitfall in the assessment of LF with BOLD imaging and native T1 mapping.

Assessment of nitrogen metabolism in rats on the background of acute toxic hepatitis and it's correction with l-arginin and l-ornitin.

The liver is the main organ responsible for the metabolism of different substances. At the same time it is the primary target organ for many toxic chemicals, which are metabolized there. Carbon tetrachloride is a wellknown hepatotoxin widely used to induce acute toxic liver injury in a wide range of laboratory animals. This substance induces oxidative damage, inflammation and fibrosis in the liver. AIM: The aim is to evaluate the peculiarities of nitrogen metabolism in rats on the background of acute toxic hepatitis and its correction with L-arginin and L-ornitin. MATERIALS AND METHODS: The study was performed on 40 outbred white male rats with experimental hepatitis, caused by carbon tetrachloride. The animals were divided into five groups: control group (the rats were simulated carbon tetrachloride poisoning and its correction by administering of olive oil and normal saline in equivalent doses), acute carbon tetrachloride hepatitis (single intraperitoneal injection of 50% carbon tetrachloride oil solution at the dose of 2 ml/kg-1 of body weight and simulation of treatment by administration of normal saline in equivalent doses), acute carbon tetrachloride hepatitis + L-ornithine (1000 mg×kg-1), acute carbon tetrachloride hepatitis + L-arginine (500 mg×kg-1) and acute carbon tetrachloride hepatitis + combination of substances. RESULTS: On the background of acute carbon tetrachloride intoxication, it was observed the development of toxic hepatitis in experimental animals, manifested by significant increasing of urea and creatinine levels in the blood serum of animals with a simultaneous decreasing of nitrite anion level. The administration of L-ornithine and L-arginine demonstrates positive impact on liver status and functions by stabilization of cell membranes and regeneration of functional capacity of injured cells. CONCLUSIONS: The results of our study confirm both the presence of unidirectional effects and absence of toxic influences of L-ornithine and L-arginine on liver cells under the conditions of acute carbon tetrachloride intoxication, which are the most important requirements for modern drugs for the treatment of hepato-renal syndrome.

Severity assessment in mice subjected to carbon tetrachloride.

The Directive 2010/63 EU requires classifying burden and severity in all procedures using laboratory animals. This study evaluated the severity of liver fibrosis induction by intraperitoneal carbon tetrachloride (CCl4) injections in mice. 29 male C57BL/6N mice were treated three times per week for 4 weeks with an intraperitoneal injection (50 µl) of either 0.6 ml/kg body weight CCl4-vehicle solution, germ oil (vehicle-control) or handling only. Severity assessment was performed using serum analysis, behavioral tests (open field test, rotarod, burrowing and nesting behavior), fecal corticosterone metabolite (FCM) measurement, and survival. The most significant group differences were noticed in the second week of treatment when the highest AST (1463 ± 1404 vs. 123.8 ± 93 U/L, p < 0.0001) and nesting values were measured. In addition, respective animals showed lower moving distances (4622 ± 1577 vs. 6157 ± 2060 cm, p < 0.01) and velocity in the Open field, identified as main factors in principal component analysis (PCA). Overall, a 50% survival rate was observed within the treatment group, in which the open field performance was a good tracer parameter for survival. In summary, this study demonstrates the feasibility of assessing severity in mice using behavioral tests and highlight the open field test as a possible threshold parameter for risk assessment of mortality.

Dynamic contrast-enhanced MRI with Gd-EOB-DTPA for the quantitative assessment of early-stage liver fibrosis induced by carbon tetrachloride in rabbits.

PURPOSE: To explore quantitative parameters obtained by dynamic contrast-enhanced magnetic resonance imaging (DCE MRI) with Gd-EOB-DTPA in discriminating early-stage liver fibrosis (LF) in a rabbit model. MATERIALS AND METHODS: LF was established in 60 rabbits by the injection of 50% CCl4 oil solution, whereas 30 rabbits served as the control group. All rabbits underwent pathological examination to determine the LF stage using the METAVIR classification system. DCE MRI was performed, and quantitative parameters, including Ktrans, Kep, Ve, Vp and Re were measured and evaluated among the different LF stages using spearman correlation coefficients and receiver operating characteristic curve. RESULTS: In all, 24, 25, and 22 rabbits had stage F0, stage F1, and stage F2 LF, respectively. Ktrans (r = 0.803) increased, and Kep (r = -0.495) and Re (r = -0.701) decreased with LF stage progression (P < 0.001), while no significant correlation was found for Ve or Vp. Ktrans and Re were significantly different between all LF stage pairs compared (F0 vs. F1, F0 vs. F2, F1 vs. F2, F0 vs. F1-F2, P < 0.05). With the exception of F0 vs. F1, Kep differed significantly between stages (P < 0.05). The AUC of Ktrans was higher than that of other quantitative parameters, with an AUC of 0.92, 0.99, 0.94 and 0.92 for staging F0 vs. F1, F0 vs. F2, F1 vs. F2, and F0 vs. F1-F2, respectively. CONCLUSION: Among quantitative parameters of Gd-EOB-DTPA DCE MRI, Ktrans was the best predictor for quantitatively differentiating early-stage LF.

Stereological assessment of nephroprotective effects of Trachyspermum ammi essential oil against carbon tetrachloride-induced nephrotoxicity in mice / Evaluación estereológica de los efectos nefroprotectores del aceite esencial de Trachyspermum ammi contra la nefrotoxicidad inducida por tetracloruro de carbono en ratones

Trachyspermum ammi (T. ammi) has been used in folk medicine as anti-inflammatory, antipyretic, antibacterial, antifungal agent. The present study was conducted to investigate the protective effect of Trachyspermum ammi (T. ammi) essential oil against CC14- induced nephrotoxicity in mice. Thirty-five mice were divided into five groups as follows; positive control received olive oil 1 mL/ kg/ip, negative control received CC14 1 mg/kg/ip + 0.5 mL distilled water orally and tree treatment groups which received CC14 similar to the negative control and 200, 800 and 1600 µg/kg of T. ammi essential oil, respectively. All treatments were done twice a week (Saturday and Wednesday) for 45 days. On the last day, blood was sampled for urea and creatinine assessment and the left kidney was removed for stereological estimations. Essential oil of T. ammi at high dose significantly (p ≤ 0.05) decreased serum levels of creatinine and urea in comparison with CC14-treated group. Total volume of the kidney, cortex, proximal convoluted tubules (PC), glomerulus, vessels and interstitial tissue as well as total length of PC and vessel were significantly (p ≤ 0.05) increased following CC14 administration and were restored toward normal levels at high dose of T. ammi. Also, high dose of T. ammi improved glomerular loss significantly (p ≤ 0.05) as compared with CC14-treated group. Due to the chemical composition of T. ammi essential oil such as tymol, p-cymene, γ-terpinene which are antioxidant, it can be concluded that the essential oil of T. ammi can ameliorated renal injury induced following CC14 toxicity via its antioxidant components.

En la medicina popular se ha utilizado el aceite esencial de Trachyspermum ammi (T. ammi) como agente antiinflamatorio, antipirético, antibacteriano y anti fúngico. El presente estudio se realizó para investigar el efecto protector de Trachyspermum ammi (T. ammi) aceite esencial contra la nefrotoxicidad inducida en ratones. Treinta y cinco ratones fueron divididos en cinco grupos de la siguiente manera; el control positivo recibió 1 mL / kg / ip de aceite de oliva, el control negativo recibió 1 mg / kg / ip + 0,5 mL de agua destilada por vía oral y grupos de tratamiento arbóreo que recibieron un control similar al negativo y 200, 800 y 1600 mg / kg de T. aceite esencial de T. ammi, respectivamente. Todos los tratamientos se realizaron dos veces por semana (sábado y miércoles) durante 45 días. En el último día de tratamiento, se tomaron muestras de sangre para evaluar la urea y la creatinina, y se extrajo el riñón izquierdo para realizar estimaciones estereológicas. El aceite esencial de T. ammi a dosis altas significativamente (p ≤ 0,05) disminuyó los niveles séricos de creatinina y urea en comparación con el grupo tratado. El volumen total del riñón, la corteza, los túbulos contorneados proximales (PC), el glomérulo, los vasos y el tejido intersticial, así como la longitud total de la PC y el vaso aumentaron significativamente (p ≤ 0,05) después de la administración y se restablecieron a niveles normales con dosis altas de T. ammi. Además, una dosis alta de T. ammi mejoró significativamente la pérdida glomerular (p ≤ 0,05) en comparación con el grupo tratado. Debido a la composición química del aceite esencial de T. ammi como timol, p-cimeno, 𝛾-terpineno con propiedades antioxidantes, se puede concluir que el aceite esencial de T. ammi puede mejorar la lesión renal inducida después de la toxicidad a través de sus componentes antioxidantes.

Conductive polymer-based bioelectrochemical assembly for in vitro cytotoxicity evaluation: Renoprotective assessment of Salvia officinalis against carbon tetrachloride induced nephrotoxicity.

BACKGROUND: The rise of organic electronics represents one of the most prominent technological developments of the last two decades, with its interface with biological systems highlighting new directions of research. The "soft" nature of conducting polymers renders them unique platforms for cell-based microdevices, allowing their implementation in drug discovery, pharmaceutical effect analysis, environmental pollutant testing etc. METHODS: Cellular adhesion, proliferation and viability experiments were carried out to verify the biocompatibility of a PEDOT conductive polymer surface. Cyclic voltammetry was employed for estimating the electrocatalytic activity of the renal cell/electrode interface. The nephrotoxicity agent CCl4 and the medicinal plant Salvia officinalis were used on the proposed assembly. Renal cell viability was also assayed through the MTT assay. RESULTS: Renal cells were able to adhere and proliferate on the conducting polymer surface. Electrochemical responses of the polymer exhibited good correlation with cell number and CCl4 concentration. Amelioration of the CCl4-induced renotoxicity by co-incubation with Salvia officinalis extract was demonstrated by both the MTT assay and the electrode's capacitance. CONCLUSIONS: A conducting polymer-based bioelectrochemical assembly was established for in vitro mammalian cytotoxicity/cytoprotection assessment, employing renal cell monolayers as the primary transducers for signal generation and biological sensing. GENERAL SIGNIFICANCE: The knowledge on PEDOT mammalian cell biocompatibility and possible applications was expanded. The proposed interdisciplinary approach connects soft electronics with biology and could provide a useful tool for preliminary crude drug screening and bioactivity studies of natural products or plant extracts in vitro.

In vitro assessment of hepatoprotective agents against damage induced by acetaminophen and CCl4.

BACKGROUND: In vitro bioassays are important in the evaluation of plants with possible hepatoprotective effects. The aims of this study were to evaluate the pretreatment of HepG2 cells with hepatoprotective agents against the damage induced by carbon tetrachloride (CCl4) and paracetamol (APAP). METHODS: Antioxidative activity was measured using an assay to measure 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging. The in vitro hepatotoxicity of CCl4 and APAP, and the cytotoxic and hepatoprotective properties of silymarin (SLM), silybinin (SLB), and silyphos (SLP) were evaluated by measuring cell viability; activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH); total antioxidant capacity (TAOxC); and reduced glutathione (GSH), superoxide dismutase (SOD), and lipid peroxidation (malondialdehyde (MDA) levels). RESULTS: Only SLB and SLM showed strong antioxidative activity in the DPPH assay (39.71 ± 0.85 µg/mL and 14.14 ± 0.65 µg/mL, respectively). CCl4 induced time- and concentration-dependent changes. CCl4 had significant effects on cell viability, enzyme activities, lipid peroxidation, TAOxC, and SOD and GSH levels. These differences remained significant up to an exposure time of 3 h. APAP induced a variety of dose- and time-dependent responses up to 72 h of exposure. SLM, SLB, and SLP were not cytotoxic. Only SLB at a concentration of 100 µg/mL or 150 µg/mL significantly decreased the enzyme activities and MDA level, and prevented depletion of total antioxidants compared with CCl4. CONCLUSIONS: CCl4 was more consistent than APAP in inducing cell injury. Only SLB provided hepatoprotection. AST, LDH, and MDA levels were good markers of liver damage.

Modernizing problem formulation for risk assessment necessitates articulation of mode of action.

The process of scientific hypothesis formulation affects the experimental designs, methods and interpretations applied, but to be testable, the hypotheses posed must conform to the state of scientific knowledge and available technology. An analogous situation exists in risk assessment, where the questions addressed are typically articulated in the problem formulation phase. Decades ago, regulatory agencies couched problem formulation according to the questions answerable by the science of the day. As regulatory requirements for risk assessment became codified, so too did the rudiments of problem formulation. Unfortunately, codifying problem formulation prevented it from evolving to keep pace with scientific advancements. Today's more advanced science is not always being used effectively and efficiently in risk assessment because the risk assessment problem formulation step still typically poses antiquated questions. Problem formulation needs to be improved so that modern science can inform risk considerations. Based on recent developments in the Human Relevance Framework and using well-studied example chemicals - chloroform and carbon tetrachloride - an approach is proposed for focusing problem formulation on human-relevant hypotheses. We contend that modernizing problem formulation in this way will make risk assessment more scientifically accurate, more practical, and more relevant for protecting human health and the environment.

Ex vivo assessment of carbon tetrachloride (CCl(4))-induced chronic injury using polarized light spectroscopy.

The liver performs various functions, such as the production and detoxification of chemicals; therefore, it is susceptible to hepatotoxins such as carbon tetrachloride (CCl4), which causes chronic injury. Thus, assessment of injury and its status of severity are of prime importance. Current work reports an ex vivo study for probing the severance of hepatic injury induced by CCl4 with polarized light over the spectral range 400-800 nm. Different concentrations of CCl4 were used to induce varying severity of hepatic injury in a rat model. Linear retardance, depolarization rates, and diagonal Mueller matrix elements (m22, m33, and m44), were successfully used as the distinguishing criterion for normal and different liver injuries. Our results show that linear retardance for injured liver samples with lower doses of CCl4 tends to increase when compared with normal liver samples, while samples injured at higher doses of CCl4 offer almost no retardance. Total, linear, and circular depolarizations follow decreasing trends with increased liver injury severity over the entire investigated wavelength range. Linear polarization states were observed to be better maintained as compared to circular polarization states for all samples. Furthermore, numerical values of diagonal elements of the experimentally measured Mueller matrix also increase with increasing doses of CCl4. Liver fibroses, change in transport albedo, and the relative refractive index of the extracellular matrix caused by CCl4 are responsible for the observed differences. These results will provide a pathway to gauge the severity of injury caused by toxic chemicals.

In vivo assessment of liver fibrosis using diffuse reflectance and fluorescence spectroscopy: a proof of concept.

A novel application of diffuse reflectance and fluorescence spectroscopy in the assessment of liver fibrosis is here reported. To induce different stages of liver fibrosis, a sufficient number of male Wistar rats were differentially exposed to chronic administration with carbon tetrachloride. Then, diffuse reflectance and fluorescence spectra were in vivo measured from the liver surface of each animal by a minimal invasive laparoscopic procedure. The liver fibrosis degree was conventionally determined by means of histological examination using the Mason's Trichrome stain, accompanied by hepatic expression of α-sma, and evaluation of the ALT/AST serum levels. The liver from rats exhibiting higher grades of fibrosis showed a significant increase in diffuse reflectance and fluorescence intensity when compared with control animals. At 365 nm, the diffuse reflectance spectrum exhibited an increase of 4 and 3-fold in mild and advanced fibrotic rats, respectively, when compared to the control group. Similarly, the fluorescence emission at 493 nm was 2-fold higher in fibrotic animals than in controls. By using fluorescence intensity, discrimination algorithms indicated 73% sensitivity and 94% specificity for recognition of hepatic fibrosis, while for diffuse reflectance, these values increased up to 85% and 100%, respectively. Taking into consideration there is a special need for developing new diagnostic approaches focused on detecting different stages of liver fibrosis with minimal invasiveness, these results suggest that diffuse reflectance and fluorescence spectroscopy could be worthy of further exploration in patients with liver disease.

A novel marker for assessment of liver matrix remodeling: an enzyme-linked immunosorbent assay (ELISA) detecting a MMP generated type I collagen neo-epitope (C1M).

A competitive enzyme-linked immunosorbent assay (ELISA) for detection of a type I collagen fragment generated by matrix metalloproteinases (MMP) -2, -9 and -13, was developed (CO1-764 or C1M). The biomarker was evaluated in two preclinical rat models of liver fibrosis: bile duct ligation (BDL) and carbon tetra chloride (CCL4)-treated rats. The assay was further evaluated in a clinical study of prostate-, lung- and breast-cancer patients stratified according to skeletal metastases. A technically robust ELISA assay specific for a MMP-2, -9 and -13 neo-epitope was produced and seen to be statistically elevated in BDL rats compared to baseline levels as well as significantly elevated in CCL4 rats stratified according to the amount of total collagen in the livers. CO1-764 levels also correlated significantly with total liver collagen and type I collagen mRNA expression in the livers. Finally, the CO1-764 marker was not correlated with skeletal involvement or number of bone metastases. This ELISA has the potential to assess the degree of liver fibrosis in a non-invasive manner.

Carbon tetrachloride marine risk assessment with special reference to the OSPARCOM region: North Sea.

This risk assessment on carbon tetrachloride was carried out specifically for the marine environment, according to the methodology laid down in the European Union (EU) risk assessment Regulation (1488/94) and the Technical Guidance Documents for New and Existing Substances (TGD, 1996). The Montreal Protocol on ozone depleting substances scheduled a phase-out of carbon tetrachloride except for some essential and feedstock uses. This study consisted of the collection and evaluation of data on effects and environmental concentrations from analytical monitoring programs in large rivers and estuaries in the North Sea area. The risk is indicated by the ratio of the Predicted Exposure Concentration (PEC) and the Predicted No-Effect Concentration (PNEC) for the marine aquatic environment. In total 16 studies for fish, 7 studies for invertebrates and 5 for algae have been evaluated. The appropriate assessment factor has been used to calculate a PNEC of 7 microg/l based on long-term exposure for three trophic levels. For coastal waters and estuaries a typical and worst case PEC of 0.003 and 0.31 microg/l are derived. For river waters a typical and worst case PEC of 0.02 and 0.12 microg/l are derived, respectively. These ratios, which do not take into account any dilution within the sea, correspond to safety margins from 25 to 2500 between the aquatic effect and the exposure concentration. Carbon tetrachloride does not meet the criteria of 'toxic, persistent and liable to bioaccumulate' substances as mentioned by the Oslo and Paris Conventions for the Prevention of Marine Pollution (OSPAR-DYNAMEC). It can be concluded that the present use of carbon tetrachloride does not present a risk to the marine aquatic environment.

The utility of PBPK in the safety assessment of chloroform and carbon tetrachloride.

Occupational exposure limits (OELs) for individual substances are established on the basis of the available toxicological information at the time of their promulgation, expert interpretation of these data in light of industrial use, and the framework in which they sit. In the United Kingdom, the establishment of specific OELs includes the application of uncertainty factors to a defined starting point, usually the NOAEL from a suitable animal study. The magnitude of the uncertainty factors is generally determined through expert judgment including a knowledge of workplace conditions and management of exposure. PBPK modeling may help in this process by informing on issues relating to extrapolation between and within species. This study was therefore designed to consider how PBPK modeling could contribute to the establishment of OELs. PBPK models were developed for chloroform (mouse and human) and carbon tetrachloride (rat and human). These substances were chosen for examination because of the extent of their toxicological databases and availability of existing PBPK models. The models were exercised to predict the rate (chloroform) or extent (carbon tetrachloride) of metabolism of these substances, in both rodents and humans. Monte Carlo analysis was used to investigate the influence of variability within the human and animal model populations. The ratio of the rates/extent of metabolism predicted for humans compared to animals was compared to the uncertainty factors involved in setting the OES. Predictions obtained from the PBPK models indicated that average rat and mouse metabolism of carbon tetrachloride and chloroform, respectively, are much greater than that of the average human. Application of Monte Carlo analysis indicated that even those people who have the fastest rates or most extensive amounts of metabolism in the population are unlikely to generate the levels of metabolite of these substances necessary to produce overt toxicity in rodents. This study highlights the value that the use of PBPK modeling may add to help inform and improve toxicological aspects of a regulatory process.

An assessment of the potential of protopine to inhibit microsomal drug metabolising enzymes and prevent chemical-induced hepatotoxicity in rodents.

The potential of protopine to inhibit microsomal drug metabolising enzymes (MDM E) and prevent paracetamol- and CCl4-induced hepatotoxicity was studied in rats. Paracetamol at the dose of 640 mg kg-1 produced hepatic damage in rats as manifested by the rise in serum levels of aspartate transaminase (AST) and alanine transaminase (ALT) to 972+/-186 and 624+/-131 IU (mean+/-sem; n=10), respectively, compared to respective control values of 101+/-29 and 64+/-18 IU. Pretreatment of rats with protopine (11 mg kg-1, orally twice daily for 2 days) lowered significantly the respective serum AST and ALT levels (P<0.05) to 289+/-52 and 178+/-43 IU. The hepatotoxic dose of CCl4 (1.5 ml kg-1; orally) raised serum AST and ALT levels to 543+/-89 and 387+/-69 IU (mean+/-sem; n=10), respectively, compared to respective control values of 98+/-28 and 56+/-17 IU. The same dose of protopine (11 mg kg-1) was able to prevent significantly (P<0.05), the CCl4-induced rise in serum enzymes and the estimated values of AST and ALT were 168+/-36 and 93+/-28 IU, respectively. Protopine caused prolongation (P<0.05) in pentobarbital (55 mg kg-1)-induced sleep as well as potentiated strychnine-induced toxicity in rats, suggestive of an inhibitory effect on MDME. These results indicate that protopine exhibits anti-hepatotoxic action which may be mediated through inhibition of MDME.

Injury and repair as opposing forces in risk assessment.

Recent advances in our understanding of the toxicodynamic events that follow infliction of injury have helped us to bridge the link between the tissue injury and the final outcome of that injury. In addition to infliction of tissue injury, toxic chemicals induce a biological compensatory response of tissue repair intended to overcome tissue injury through healing. Since stimulation of tissue repair is a simultaneous response accompanying injury, measuring this response in addition to quantifying injury might be helpful in tomorrow's risk assessment. Studies with model hepatotoxicants such as thioacetamide and CCl4, where tissue repair as well as injury were measured, reveal that endogenous mechanisms that drive the tissue repair response are responsible for more than just compensation for tissue injury. Up to a threshold dose, tissue repair is stimulated in a dose-dependent manner, and above this threshold it is both delayed and diminished. During this delay, tissue injury progresses unabated leading to tissue destruction and animal death. While dose-related stimulation of tissue repair leads to recovery, delayed and diminished tissue repair seen at the high doses leads to tissue destruction and animal death. These findings impact on the currently used maximum tolerated doses (MTDs) in cancer bioassays. MTDs represent maximal stimulation of cell proliferation thereby enhancing the likelihood of errors in DNA replication. Measuring tissue repair and injury as simultaneous biological responses to toxic agents might increase the usefulness of dose-response paradigms in risk assessment.

Physiologically based pharmacokinetic/pharmacodynamic modeling of chemical mixtures and possible applications in risk assessment.

Human exposure to chemicals, be it environmental or occupational, is rarely, if ever, limited to a single chemical. Therefore, it is essential that we consider multiple chemical effects and interactions in our risk assessment process. However, with the almost infinitely large number of chemical mixtures in the environment, systematic studies of the toxicology of these chemical mixtures with conventional methodologies and approaches are impossible because of the immense resources and unrealistically long durations required. Thus, the development of predictive and alternative toxicology method is imperative. In order to have a reasonable chance to deal with the complex issue of toxicology of chemical mixtures, we believe that the following concepts must be considered: (1) the exploitation of recent advances in computational technology; (2) the utilization of mathematical/statistical modeling; (3) coupling computer modeling with very focused, mechanistically based, and short-term toxicology studies. Our approach is, therefore, the utilization of physiologically based pharmacokinetic/pharmacodynamic (PB-PK/PD) modeling, coupled with very focused, model-directed toxicology experiments as well as other statistical/mathematical modeling such as isobolographic analysis and response surface methodology. Tissue dosimetry at the pharmacokinetic and pharmacodynamic levels is achievable with simple and complex, but chemically defined, mixtures. Our long-term goal is to formulate innovative risk assessment methodologies for chemical mixtures. In this presentation, one of our specific research projects is described: PB-PK/PD modeling of toxicologic interactions between Kepone and carbon tetrachloride (CCl4) and the coupling of Monte Carlo simulation for the prediction of acute toxicity.

Toxicology studies of a chemical mixture of 25 groundwater contaminants: hepatic and renal assessment, response to carbon tetrachloride challenge, and influence of treatment-induced water restriction.

Because groundwater contamination is an important environmental concern, we examined the hepatic and renal effects of repeated exposure to a mixture of 25 chemicals frequently found in groundwater near hazardous-waste disposal sites and the effect of such exposure on carbon tetrachloride (CCI4) toxicity. Adult male F-344 rats received ad libitum deionized water and feed (Ad Lib Water) or ad libitum 10% MIX (referring to 10% of a technically achievable stock mixture) and feed for 14 d. Because exposure to the 25-chemical mixture via the drinking water resulted in decreased water and feed consumption, restricted deionized water and feed controls (Restricted Water) were included. On d 14, rats were gavaged with 0, 0.0375, 0.05, 0.075 or 0.15 ml CCl4/kg, and hepatic and renal toxicity assessed 24 h later. Little or no hepatic and renal toxicity was observed in rats exposed to 10% MIX alone. No hepatic or renal lesions occurred that could be attributed to 10% MIX alone. Slight but statistically significant alterations, of uncertain biological significance, resulted from the water treatments: 10% MIX increased alanine aminotransferase, urea nitrogen (BUN), and BUN/creatinine ratio; Restricted Water increased 5'-nucleotidase and decreased alkaline phosphatase. Relative kidney weight was increased by both 10% MIX and Restricted Water. CCI4 resulted in significant dosage-dependent hepatotoxicity in all three water treatment groups but had little or no effect on renal indicators of toxicity. Relative to Ad Lib Water, significantly greater hepatotoxicity occurred in both 10% MIX and Restricted Water rats. The response to CCI4 in the Restricted Water rats was similar to that of 10% MIX rats, indicating that a substantial portion of the effect of 10% MIX on CCI4 hepatotoxicity is due to decreased water and feed intake.

Biologically based modeling in toxicology research.

Biologically based modeling can be described as the process by which the specific mechanistic steps governing tissue disposition and toxic action of chemicals are expressed in quantitative terms by a set of equations leading to prediction of the outcome of specific toxicological experiments by computer simulation. These models are useful in risk assessment because their mechanistic biological basis permits the high-to-low dose, route to route and interspecies extrapolation of the tissue disposition and toxic action of chemicals. By far their greatest utility is not as "finished" risk assessment models, but as research tools that convey a quantitative expression of our hypotheses of tissue disposition and toxic action of a chemical. A structured modeling approach to toxicology problems helps identify the data gaps in the areas of chemical disposition and toxic action, thus prioritizing on-going research to obtain critical information required to conduct quantitative risk assessment. This paper examines progress in developing comprehensive biologically based models for cancer induction by non-genotoxic carcinogens that are cytotoxic in target tissues. The strategies for linking the models on dosimetry, cytotoxicity, and carcinogenicity are described in detail. The basic concepts and approaches discussed here can be applied to many other toxic chemicals and to toxicity endpoints other than cancer.