Evaluation of the genotoxic potential of single-wall carbon nanotubes by using a battery of in vitro and in vivo genotoxicity assays

The genotoxic potential of a high purity sample of single-wall carbon nanotubes (SWCNTs) was evaluated using a battery of in vitro and in vivo genotoxicity assays. These comprised a bacterial reverse mutation test (Ames test), an in vitro chromosomal aberration test, and an in vivo mouse bone marrow micronucleus test. The SWCNTs exerted no genotoxicity in Salmonella typhimurium TA97, TA98, TA100, and TA1535, or in Escherichia coli WP2 uvrA/pKM101, whether in the absence or presence of metabolic activation and at concentrations of 12.5–500 μg/plate. In the chromosomal aberration test, at 300–1000 μg/mL, the SWCNTs did not increase the number of structural or numerical chromosomal aberrations, whether the test was conducted with or without metabolic activation. In the in vivo bone marrow micronucleus test, doses of 60 mg/kg and 200 mg/kg SWCNTs did not affect the proportions of immature and total erythrocytes, nor did it increase the number of micronuclei in the immature erythrocytes of mice. The results of these studies show that the high purity and well-dispersed sample of SWCNTs are not genotoxic under the conditions of the in vitro bacterial reverse mutation assay, chromosomal aberration assay, or in vivo bone marrow micronucleus test, and thus appear not to pose a genotoxic risk to human health in vivo.     

Integrating in vitro testing and physiologically-based pharmacokinetic (PBPK) modelling for chemical liver toxicity assessment—A case study of troglitazone

In vitro to in vivo extrapolation (IVIVE) for next-generation risk assessment (NGRA) of chemicals requires computational modeling and faces unique challenges. Using mitochondria-related toxicity data of troglitazone (TGZ), a prototype drug known for liver toxicity, from HepaRG, HepG2, HC-04, and primary human hepatocytes, we explored inherent uncertainties in IVIVE, including cell models, cellular response endpoints, and dose metrics. A human population physiologically-based pharmacokinetic (PBPK) model for TGZ was developed to predict in vivo doses from in vitro point-of-departure (POD) concentrations. Compared to the 200–800 mg/d dose range of TGZ where liver injury was observed clinically, the predicted POD doses for the mean and top one percentile of the PBPK population were 28–372 and 15–178 mg/d respectively based on C_max dosimetry, and 185–2552 and 83–1010 mg/d respectively based on AUC. In conclusion, although with many uncertainties, integrating in vitro assays and PBPK modeling is promising in informing liver toxicity-inducing TGZ doses.     

三维重组皮肤模型在体外替代遗传毒性评价中的应用

三维（3D）重组皮肤模型已证实在模拟体内代谢条件、给药浓度及反应靶器官毒性特点方面具有突出优势。近年来已有多家公司构建3D重组人工皮肤模型,且一些制药公司已将3D细胞模型应用于药物的早期毒性筛选。我国动物实验替代方法的研究仍处于起步阶段,利用3D重组皮肤模型进行体外安全性评价成为目前替代方法的研究热点之一。综述人3D重组皮肤模型在遗传毒性评价中体外微核试验和彗星试验的研究进展,并对该模型在外用药物体外替代遗传毒性评价中的应用前景进行探讨。     

Evaluation of Possible Genotoxic Mechanisms for Acrylonitrile Tumorigenicity

Acrylonitrile (ACN) exposure is associated with tumors in rat brain, Zymbal gland, and mammary gland. Adducts affecting base pairing were formed in isolated DNA exposedin vitroto the ACN metabolite cyanoethylene oxide (CNEO). DNA from liver, which is not a cancer target organ in ACN-exposed rats, contained low levels of 7-(2-oxoethyl)guanine, an adduct believed not to interfere with base pairing. No adducts have been detected in brain DNA from ACN-exposed rats, suggesting that brain tumors may have arisen by mechanisms other than ACN–DNA reactivity. Genotoxicity assays of ACN have indicated no particular carcinogenic mechanism. Positive reverse mutagenesis inSalmonella typhimuriumHisG46 base substitution tester strains by ACN is attributable to CNEO. Otherin vitrogenotoxicity test assays of ACN have yielded mixed results, without consistent effect of metabolic activation. Some positive genotoxicity data for ACN appear to result from artifacts or from non-DNA-reactive mechanisms.In vivomicronucleus, chromosome aberration, and autoradiographic unscheduled DNA synthesis assays were negative for ACN. The comparative genotoxicity of vinyl chloride and ACN indicates that despite other similarities, they cause rodent tumors by different mechanisms. Also, the absence of ACN–DNA adduct formation in the rat brain suggests the operation of epigenetic mechanisms.     

Biotransformation of cyclosporin in primary rat,porcine and human liver cell co-cultures

The purpose of this study was to investigate the species-specific cyclosporin biotransformation in primary rat, human, and porcine liver cell cultures, and to investigate the suitability of a modified sandwich culture technique with non-purified liver cell co-cultures for drug metabolism studies. A sandwich culture was found to enhance hepatocellular metabolic activity and improve cellular morphology and ultrastructure. The cyclosporin metabolites AM9 and AM1 were formed in porcine and human liver cell sandwich co-cultures at levels corresponding to the respective in vivo situations. In contrast, metabolite profiles in rat hepatocytes were at variance with the in vivo situation. However, for all cell types, the overall metabolic activity was positively influenced by sandwich co-culture. The initial levels of albumin synthesis were higher in sandwich cultures than in those without matrix overlay. It is hypothesized that the sandwich culture system provides an improved microenvironment and is, therefore, an advantageous tool for in vitro studies of drug metabolism.     

Recent advances in three-dimensional cell culturing to assess liver function and dysfunction: from a drug biotransformation and toxicity perspective

The liver is a vital organ fulfilling a central role in over 500 major metabolic functions, including serving as the most essential site for drug biotransformation. Dysfunction of the drug biotransformation processes may result in the exposure of the liver (and other organs) to hepatotoxins, potentially interacting with cellular constituents and causing toxicity and various lesions. Hepatotoxicity can be investigated on a tissue, cellular and molecular level by employing various in vivo and in vitro techniques, including novel three-dimensional (3?D) cell culturing methods. This paper reflects on the liver and its myriad of functions and the influence of drug biotransformation on liver dysfunction. Current in vivo and in vitro models used to study liver function and dysfunction is outlined, emphasizing their advantages and disadvantages. The advantages of novel in vitro 3?D cell culture models are discussed and the possibility of novel models to bridge the gap between in vitro and in vivo models is explained. Progression made in the field of cell culturing methods such as 3?D cell culturing techniques over the last decade promises to reduce the use of in vivo animal models in biotransformation and toxicological studies of the liver.     

An in vivo study of nanorod,nanosphere, and nanowire forms of titanium dioxide using Drosophila melanogaster: toxicity,cellular uptake,oxidative stress,and DNA damage

ABSTRACT

The biological impact of nanomaterials (NMs) is determined by several factors such as size and shape, which need to be taken into consideration in any type of analysis. While investigators often prefer to conduct in vitro studies for detection of any possible adverse effects of NMs, in vivo approaches yield more relevant data for risk assessment. For this reason, Drosophila melanogaster was selected as a suitable in vivo model to characterize the potential risks associated with exposure nanorods (NRs), nanospheres (NSs), nanowires (NWs) forms of titanium dioxide (TiO₂), and their microparticulated (or bulk) form, as TiO₂. Third instar larvae (72 hr old larvae) were fed with TiO₂ (NRs, NSs, or NWs) and TiO₂ at concentrations ranging from 0.01 to 10 mM. Viability (toxicity), internalization (cellular uptake), intracellular reactive oxygen species (ROS) production, and genotoxicity (Comet assay) were the end-points evaluated in hemocyte D. melanogaster larvae. Significant intracellular oxidative stress and genotoxicity were noted at the highest exposure concentration (10 mM) of TiO₂ (NRs, NSs, or NWs), as determined by the Comet assay and ROS analysis, respectively. A concentration–effect relationship was observed in hemocytes exposed to the NMs. Data demonstrated that selected forms of TiO₂.-induced genotoxicity in D. melanogaster larvae hemocytes indicating this organism is susceptible for use as a model to examine in vivo NMs-mediated effects.     

In vivo and in vitro investigations on biological effects of aromatic bis-(2-chloroethyl)amino-bisphosphonic acids,new agents proposed for chemotherapy of bone tumors: cytostatic activity in rat osteosarcoma; toxicity and genotoxicity in liver and bone marrow; mutagenicity in S. Typhimurium

Summary Two new aromatic bis-(2-chloroethyl)-amino derivatives (BCMP and BAD) which are linked to osteotropic bisphosphonates were investigated for their therapeutical efficacy in rat osteosarcoma. Furthermore their genotoxic potential in vitro was determined in S. typhimurium and in mammalian cells. Finally, parameters for toxicity and genotoxicity were determined in liver and bone marrow cells following in vivo treatment. It was shown that BAD was of higher therapeutic effectiveness than BCMP. Both compounds induced approximately a two fold increase of his⁺ revertants in S. typhimurium TA1535 following metabolic activation by subcellular liver fractions. Both compounds also induced amplification of SV40 DNA in SV40 transformed cells (CO631). This endpoint may be of importance for acquired resistancy of cells during therapy. DNA-single strand breaks were induced by BCMP but not by BAD in liver cells and CO631 cell line. Following in vivo treatment BCMP was of higher genotoxic activity in liver cells than BAD. In comparison, genotoxicity of both compounds was much lower in bone marrow cells than in liver cells. BCMP was again more potent than BAD in inducing DNA single strand breaks, whereas BAD was more toxic. The higher therapeutic efficacy of BAD together with its lower genotoxic properties makes this compound superior to BCMP as a candidate for applied chemotherapy in humans.     

The Use of Human Hepatocytes to Select Compounds Based on Their Expected Hepatic Extraction Ratios in Humans

Purpose. The present investigation retrospectively evaluates the use of human hepatocytes to classify compounds into low, intermediate or high hepatic extraction ratio in man. Methods. A simple approach was used to correlate the in vivo hepatic extraction ratio of a number of compounds in man (literature and in-house data) with the corresponding in vitro clearance which was determined in human hepatocytes. The present approach assumes that, for compounds eliminated mainly through liver metabolism, intrinsic clearance is the major determinant for their in vivo hepatic extraction ratio and subsequently their bioavailability in man. The test compounds were selected to represent a broad range of extraction ratios and a variety of metabolic pathways. Results. The present data show that in vitro clearances in human hepatocytes are predictive for the hepatic extraction ratios in vivo in man. Most of the test compounds (n = 19) were successfully classified based upon human hepatocyte data into low, intermediate or high hepatic extraction compounds, i.e. compounds with potential for high, intermediate or low bioavailabilities in humans. Conclusions. The present approach, validated so far with 19 test compounds, appears to be a valuable tool to screen for compounds with respect to liver first-pass metabolism at an early phase of drug discovery.     

The Cosmetics Europe strategy for animal-free genotoxicity testing: Project status up-date

The Cosmetics Europe (formerly COLIPA) Genotoxicity Task Force has driven and funded three projects to help address the high rate of misleading positives in in vitro genotoxicity tests:The completed “False Positives” project optimized current mammalian cell assays and showed that the predictive capacity of the in vitro micronucleus assay was improved dramatically by selecting more relevant cells and more sensitive toxicity measures.The on-going “3D skin model” project has been developed and is now validating the use of human reconstructed skin (RS) models in combination with the micronucleus (MN) and Comet assays. These models better reflect the in use conditions of dermally applied products, such as cosmetics. Both assays have demonstrated good inter- and intra-laboratory reproducibility and are entering validation stages.The completed “Metabolism” project investigated enzyme capacities of human skin and RS models. The RS models were shown to have comparable metabolic capacity to native human skin, confirming their usefulness for testing of compounds with dermal exposure.The program has already helped to improve the initial test battery predictivity and the RS projects have provided sound support for their use as a follow-up test in the assessment of the genotoxic hazard of cosmetic ingredients in the absence of in vivo data.     

Prediction of Hepatic Clearance in Human From In Vitro Data for Successful Drug Development

The in vivo metabolic clearance in human has been successfully predicted by using in vitro data of metabolic stability in cryopreserved preparations of human hepatocytes. In the predictions by human hepatocytes, the systematic underpredictions of in vivo clearance have been commonly observed among different datasets. The regression-based scaling factor for the in vitro-to-in vivo extrapolation has mitigated discrepancy between in vitro prediction and in vivo observation. In addition to the elimination by metabolic degradation, the important roles of transporter-mediated hepatic uptake and canalicular excretion have been increasingly recognized as a rate-determining step in the hepatic clearance. It has been, therefore, proposed that the in vitro assessment should allow the evaluation of clearances for both transporter(s)-mediated uptake/excretion and metabolic degradation. This review first outlines the limited ability of subcellular fractions such as liver microsomes to predict hepatic clearance in vivo. It highlights the advantages of cryopreserved human hepatocytes as one of the versatile in vitro systems for the prediction of in vivo metabolic clearance in human at the early development stage. The following section discusses the mechanisms underlying the systematic underprediction of in vivo intrinsic clearance by hepatocytes. It leads to the proposal for the assessment of hepatic uptake clearance as one of the kinetically important determinants for accurate predictions of hepatic clearance in human. The judicious combination of advanced technologies and understandings for the drug disposition allows us to rationally optimize new chemical entities to the drug candidate with higher probability of success during the clinical development.     

Chimeric mice transplanted with human hepatocytes as a model for prediction of human drug metabolism and pharmacokinetics

Preclinical studies in animal models are used routinely during drug development, but species differences of pharmacokinetics (PK) between animals and humans have to be taken into account in interpreting the results. Human hepatocytes are also widely used to examine metabolic activities mediated by cytochrome P450 (P450) and other enzymes, but such in vitro metabolic studies also have limitations. Recently, chimeric mice with humanized liver (h‐chimeric mice), generated by transplantation of human donor hepatocytes, have been developed as a model for the prediction of metabolism and PK in humans, using both in vitro and in vivo approaches. The expression of human‐specific metabolic enzymes and metabolic activities was confirmed in humanized liver of h‐chimeric mice with high replacement ratios, and several reports indicate that the profiles of P450 and non‐P450 metabolism in these mice adequately reflect those in humans. Further, the combined use of h‐chimeric mice and r‐chimeric mice, in which endogenous hepatocytes are replaced with rat hepatocytes, is a promising approach for evaluation of species differences in drug metabolism. Recent work has shown that data obtained in h‐chimeric mice enable the semi‐quantitative prediction of not only metabolites, but also PK parameters, such as hepatic clearance, of drug candidates in humans, although some limitations remain because of differences in the metabolic activities, hepatic blood flow and liver structure between humans and mice. In addition, fresh h‐hepatocytes can be isolated reproducibly from h‐chimeric mice for metabolic studies. Copyright © 2013 John Wiley & Sons, Ltd.     

The regulation of human hepatic drug transporter expression by activation of xenobiotic-sensing nuclear receptors

Introduction: If a drug is found to be an inducer of hepatic drug metabolizing enzymes via activation of nuclear receptors such as pregnane X receptor (PXR) or constitutive androstane receptor (CAR), it is likely that drug transporters regulated through these same receptors will be induced as well. This review highlights what is currently known about the molecular mechanisms that regulate transporter expression and where the research is directed.

Areas covered: This review is focused on publications that describe the role of activated hepatic nuclear receptors in the subsequent regulation of drug uptake and/or efflux transporters following exposure to xenobiotics.

Expert opinion: Many of the published studies on the role of nuclear receptors in the regulation of drug transporters involve non-human test animals. But due to species response differences, these associations are not always applicable to humans. For this reason, some relevant human in vitro models have been developed, such as primary or cryopreserved human hepatocytes, human liver slices, or HepG2 or HuH7 cell lines transiently or stably transfected with PXR expression and reporter constructs as well as in vivo models such as PXR-humanized mice. These human-relevant test systems will continue to be developed and applied for the testing of investigational drugs.     

Qualitative and quantitative prediction of human in vivo metabolic pathways in a human hepatocyte-murine stromal cell co-culture model

Abstract

1.?This study assessed the value of a static in vitro human hepatocyte-murine stromal cell co-culture model to qualitatively and quantitatively predict human in vivo metabolic clearance pathways using ¹⁴C-labeled test compounds and compared these results to an in vitro suspended human hepatocyte model and the in vivo human ¹⁴C ADME studies.

2.?Test compounds represented a diverse set of clearance pathways (Phase I and Phase II). Compounds were incubated for 4?h in suspended human hepatocytes and for 24 and 168?h in the human co-culture model. Multivariate analysis revealed that long-term (168?h) incubation of test compounds in the co-culture had reasonable quantitative prediction of the in vivo human clearance pathways as compared to the 4?h suspended hepatocytes or the 24?h co-culture incubation.

3.?In vivo and in vitro disconnects were observed in cases where extra-hepatic metabolism or urinary excretion was observed in vivo. Differences in the relative percentages of Phase I and Phase II metabolites observed were likely due to microbial β-glucuronidase hydrolysis of conjugates and microflora mediated metabolism in the gut not present in the in vitro systems.     

Utility of models of the gastrointestinal tract for assessment of the digestion and absorption of engineered nanomaterials released from food matrices

Abstract

Engineered metal/mineral, lipid and biochemical macromolecule nanomaterials (NMs) have potential applications in food. Methodologies for the assessment of NM digestion and bioavailability in the gastrointestinal tract are nascent and require refinement. A working group was tasked by the International Life Sciences Institute NanoRelease Food Additive project to review existing models of the gastrointestinal tract in health and disease, and the utility of these models for the assessment of the uptake of NMs intended for food. Gastrointestinal digestion and absorption could be addressed in a tiered approach using in silico computational models, in vitro non-cellular fluid systems and in vitro cell culture models, after which the necessity of ex vivo organ culture and in vivo animal studies can be considered. Examples of NM quantification in gastrointestinal tract fluids and tissues are emerging; however, few standardized analytical techniques are available. Coupling of these techniques to gastrointestinal models, along with further standardization, will further strengthen methodologies for risk assessment.     

Bis(hydroxyphenyl)methane—bisphenol F—metabolism by the HepG2 human hepatoma cell line and cryopreserved human hepatocytes

Bisphenol F (BPF) is present in the environment and as a contaminant of food. Humans may, therefore, be exposed to BPF, and an assessment of this risk is required. BPF has been shown to have genotoxic and endocrine-disruptor properties in a human hepatoma cell line (HepG2), which is a model system for studies of xenobiotic toxicity. In this study, we investigated the ability of HepG2 cells to biotransform BPF, because metabolism may affect the observed effects of BPF, and we compared this metabolic capacity with that of human hepatocytes. Cells were incubated for 24 hours with [³H]-BPF. The culture medium was then concentrated and its metabolites were isolated by high-performance liquid chromatography and identified by mass spectrometry. BPF was largely metabolized into the corresponding sulfate by the HepG2 cell line. BPF was metabolized into both sulfate and glucuronide by human hepatocytes, but with differences between individuals. The metabolism of BPF in both HepG2 cells and human hepatocytes suggests the existence of a detoxification pathway. Thus, these two cell models differ in metabolic capacity. It is, therefore, very important, when assessing the toxic effects of substances in vitro, to determine, in parallel, the biotransformation capacities of the model used to extrapolate in vivo.     

Lack of in vitro-in vivo correlation of a novel investigational anticancer agent,SH 30

In solid tumors, the reasons for the lack of in vitro andin vivo correlation of drugactivities are multifold and includespermeability to the tumor cells,interstitial hypertension and metabolicdegradation. So, it is important to studythe permeability and metabolic dispositionof new compounds early in discovery anddevelopment of anticancer drugs. Anexperimental anti-cancer drug, SH 30demonstrated highly selective and potentcytotoxic activity against a number ofmulti-drug resistant tumor cell lines in vitro. However, it was inactive in amurine tumor model. This study wasconducted to identify the barriers thatresult in lack of correlation between in vitro and in vivo cytotoxicactivity of novel anticancer agents. Twoimportant barriers: physical(permeability) and metabolic (enzymaticinactivation) to poor delivery of SH 30 tosolid tumors were investigated in thisstudy. Tumors were sliced to separate thevascular and avascular sections. Theconcentrations of the drug at variousregions of the tumor after single andmultiple doses were investigated todetermine the permeability barrier. Thepermeability barrier was also probed usingtwo in vitro model systems, namely,matrigel® films representing extracellularmatrix and caco-2 multilayer cell culturesthat simulate solid tumors. The drug andits metabolite concentrations weredetermined in the plasma and tumors todetermine the metabolic barrier to the drugcytotoxic action. The metabolic barrierwas further probed using in vitromouse hepatocytes and liver microsomepreparations. Our examination revealed themetabolic barrier to be the majorcontributor to the ineffectiveness of SH 30in vivo. Examination ofconcentration of the drug across variousregions of the tumor corroborated by datafrom in vitro permeation studiessuggested that, for SH 30, permeabilitybarrier did not exist. After singleinjection, the concentrations of SH 30 andits metabolites in plasma and tumor werecomparable to another investigational drugwith similar features (XK 469). Contraryto day 1, after 8 consecutive days ofadministration, SH 30 concentrations weresignificantly lower, while the metabolitesconcentrations were higher, suggestingextensive metabolism due to induction ofenzyme(s). The in vitro hepatocytesand liver microsome results also showed SH30 biotransformation to the samemetabolites. Neither drug penetration, nordrug distribution into regions of thetumors distal to vasculature were impeded. The inactivity of SH 30 in vivo isprimarily due to induction of extensivemetabolism to inactive metabolites. Thismetabolism prevents adequate drug levelsbeing achieved in the tumor.     

Chimeric mice with humanized liver: Application in drug metabolism and pharmacokinetics studies for drug discovery

Predicting human drug metabolism and pharmacokinetics (PK) is key to drug discovery. In particular, it is important to predict human PK, metabolite profiles and drug-drug interactions (DDIs). Various methods have been used for such predictions, including in vitro metabolic studies using human biological samples, such as hepatic microsomes and hepatocytes, and in vivo studies using experimental animals. However, prediction studies using these methods are often inconclusive due to discrepancies between in vitro and in vivo results, and interspecies differences in drug metabolism. Further, the prediction methods have changed from qualitative to quantitative to solve these issues. Chimeric mice with humanized liver have been developed, in which mouse liver cells are mostly replaced with human hepatocytes. Since human drug metabolizing enzymes are expressed in the liver of these mice, they are regarded as suitable models for mimicking the drug metabolism and PK observed in humans; therefore, these mice are useful for predicting human drug metabolism and PK. In this review, we discuss the current state, issues, and future directions of predicting human drug metabolism and PK using chimeric mice with humanized liver in drug discovery.     

Genotoxicity studies on HM10760A,recombinant human erythropoietin conjugated to globin fragment

HM10760A is a recombinant human erythropoietin chemically conjugated to the N-terminus of human immunoglobulin Fc fragment through a polyethylene glycol linker. HM10760A was shown to have a relatively long half-life, compared with unconjugated recombinant erythropoietin. In this study, the genotoxicity of HM10760A was investigated by using a test battery of three different methods. In the Ames assay, five strains (TA100, TA1535, TA98, TA1537, and Escherichia coli WP2 uvrA) were tested at six concentrations of 3.13, 6.25, 12.5, 25, 50, and 100?μg/plate. HM10760A did not increase the number of revertant colonies in any tester strains with and without metabolic activation by rat-liver S9 mix. Subsequently, in vitro chromosomal aberration test, using Chinese hamster lung cells, were conducted at the concentrations of 25, 50, and 100?μg/mL. HM10760A did not induce chromosomal aberrations either in the short-period (6 hours) test with or without rat-liver S9 mix or in the continuous-treatment (24 hours) test. In the in vivo bone marrow micronucleus assay using the male ICR (imprinting control region) mouse, HM10760A was subcutaneously administered twice at 24-hour intervals at doses of 0, 150, 300, and 600?μg/kg. HM10760A produced a slight, but statistically significant, increase in the frequency of micronucleated polychromatic erythrocytes at 600?μg/kg. However, no biological significance was assumed, because this value was within the historical control range. From these findings obtained from the genotoxicity assays performed in this study, it appears unlikely that HM10760A acts as a genotoxic agent in vitro and in vivo.     

Mechanism of Protection Against Carbon Tetrachloride Toxicity. I. Prevention of Lethal Effects by Partial Surgical Hepatectomy

ABSTRACT

Both partial surgical hepatectomy and a challenge with a small dose of CC₁₄ depress the metabolism of xenobiotics in the liver. Infact, hepatocytes become provided with metabolic activity rdtes which are peculiar of either embryo or newborn rat lver.

These experiments have shown that partial surgical hepatectomy prevents rats from death caused by otherwise lethal doses of CC₁₄. At the same time, sham-operated animals survive to a limited ex= tent after a large dose of the halogenocompound.

Investigations carried out on the metabolic efficiency of liver microsomes, both in vitro and in vivo, clearly demonstrate that the preventive effect against CC₁₄ depends mainly on the impaired metabolic activity of endoplasmic reticulum.