Human and animal hepatocytes in vitro with extrapolation in vivo

Human and animal hepatocytes are now being used as an in vitro technique to aid drug discovery by predicting the in vivo metabolic pathways of drugs or new chemical entities (NCEs), identifying drug-metabolizing enzymes and predicting their in vivo induction. Because of the difficulty of establishing whether the cytotoxic susceptibility of human hepatocytes to xenobiotics/drugs in vitro could be used to predict in vivo human hepatotoxicity, a comparison of the susceptibility of the hepatocytes of human and animal models to six chemical classes of drugs/xenobiotics in vitro have been related to their in vivo hepatotoxicity and the corresponding activity of their metabolizing enzymes. This study showed that the cytotoxic effectiveness of 16 halobenzenes towards rat hepatocytes in vitro using higher doses and short incubation times correlated well with rat hepatotoxic effectiveness in vivo with lower doses/longer times. The hepatic/hepatocyte xenobiotic metabolizing enzyme activities of various animal species and human have been reviewed for use by veterinarians and research scientists. Where possible, recommendations have been made regarding which animal hepatocyte model is most applicable for modeling the susceptibility to xenobiotic induced hepatotoxicity of those humans with slow versus rapid metabolizing enzyme polymorphisms. These recommendations are based on the best human fit for animal drug/xenobiotic metabolizing enzymes in terms of activity, kinetics and substrate/inhibitor specificity. The use of human hepatocytes from slow versus rapid metabolizing individuals for drug metabolism/cytotoxicity studies; and the research use of freshly isolated rat hepatocytes and "Accelerated Cytotoxicity Mechanism Screening" (ACMS) techniques for identifying drug/xenobiotic reactive metabolites are also described. Using these techniques the molecular hepatocytotoxic mechanisms found in vitro for seven classes of xenobiotics/drugs were found to be similar to the rat hepatotoxic mechanisms reported in vivo.     

Cholesterol-Secreting and Statin-Responsive Hepatocytes from Human ES and iPS Cells to Model Hepatic Involvement in Cardiovascular Health

Hepatocytes play a central and crucial role in cholesterol and lipid homeostasis, and their proper function is of key importance for cardiovascular health. In particular, hepatocytes (especially periportal hepatocytes) endogenously synthesize large amounts of cholesterol and secrete it into circulating blood via apolipoprotein particles. Cholesterol-secreting hepatocytes are also the clinically-relevant cells targeted by statin treatment in vivo. The study of cholesterol homeostasis is largely restricted to the use of animal models and immortalized cell lines that do not recapitulate those key aspects of normal human hepatocyte function that result from genetic variation of individuals within a population. Hepatocyte-like cells (HLCs) derived from human embryonic and induced pluripotent stem cells can provide a cell culture model for the study of cholesterol homeostasis, dyslipidemias, the action of statins and other pharmaceuticals important for cardiovascular health. We have analyzed expression of core components for cholesterol homeostasis in untreated human iPS cells and in response to pravastatin. Here we show the production of differentiated cells resembling periportal hepatocytes from human pluripotent stem cells. These cells express a broad range of apolipoproteins required for secretion and elimination of serum cholesterol, actively secrete cholesterol into the medium, and respond functionally to statin treatment by reduced cholesterol secretion. Our research shows that HLCs derived from human pluripotent cells provide a robust cell culture system for the investigation of the hepatic contribution to human cholesterol homeostasis at both cellular and molecular levels. Importantly, it permits for the first time to also functionally assess the impact of genetic polymorphisms on cholesterol homeostasis. Finally, the system will also be useful for mechanistic studies of heritable dyslipidemias, drug discovery, and investigation of modes of action of cholesterol-modulatory drugs.     

Cryopreservation of isolated human hepatocytes for transplantation: State of the art

Hepatocytes isolated from unused donor livers are being used for transplantation in patients with acute liver failure and liver-based metabolic defects. As large numbers of hepatocytes can be prepared from a single liver and hepatocytes need to be available for emergency and repeated treatment of patients it is essential to be able to cryopreserve and store cells with good thawed cell function. This review considers the current status of cryopreservation of human hepatocytes discussing the different stages involved in the process. These include pre-treatment of cells, freezing solution, cryoprotectants and freezing and thawing protocols. There are detrimental effects of cryopreservation on hepatocyte structure and metabolic function, including cell attachment, which is important to the engraftment of transplanted cells in the liver. Cryopreserved human hepatocytes have been successfully used in clinical transplantation, with evidence of replacement of missing function. Further optimisation of hepatocyte cryopreservation protocols is important for their use in hepatocyte transplantation.     

Human hepatocytes: isolation, cryopreservation and applications in drug development

The recent developments in the isolation, culturing, and cryopreservation of human hepatocytes, and the application of the cells in drug development are reviewed. Recent advances include the improvement of cryopreservation procedures to allow cell attachment, thereby extending the use of the cells to assays that requires prolong culturing such as enzyme induction studies. Applications of human hepatocytes in drug development include the evaluation of metabolic stability, metabolite profiling and identification, drug-drug interaction potential, and hepatotoxic potential. The use of intact human hepatocytes, because of the complete, undisrupted metabolic pathways and cofactors, allows the development of data more relevant to humans in vivo than tissue fractions such as human liver microsomes. Incorporation of key in vivo factors with the intact hepatocytes in vitro may help predictive human in vivo drug properties. For instance, evaluation of drug metabolism and drug-drug interactions with intact human hepatocytes in 100% human serum may eliminate the need to determine in vivo intracellular concentrations for the extrapolation of in vitro data to in vivo. Co-culturing of hepatocytes and nonhepatic primary cells from other organs in the integrated discrete multiple organ co-culture (IdMOC) may allow the evaluation of multiple organ interactions in drug metabolism and drug toxicity. In conclusion, human hepatocytes represent a critical experimental model for drug development, allowing early evaluation of human drug properties to guide the design and selection of drug candidates with a high probability of clinical success.     

Investigation of Functional and Morphological Integrity of Freshly Isolated and Cryopreserved Human Hepatocytes

There is a pressing need for alternative therapeutic methods effective in the treatment of patients with liver insufficiency. Isolated human hepatocytes may be a viable alternative or adjunct to orthotopic liver transplantation in such patients. The purpose of this study was to evaluate the viability and functional integrity of freshly isolated and cryopreserved human hepatocytes, in preparation for a multi-center human hepatocyte transplantation trial. We are currently processing transplant-grade human parenchymal liver cells from nondiseased human livers that are obtained through a network of organ procurement organizations (OPOs). Thus far, sixteen hepatocyte transplants have been performed using hepatocytes processed by our methods. At the time of referral all specimens were deemed unsuitable for transplantation due to anatomical anomalies, high fat content, medical history, etc. Hepatocytes were isolated from encapsulated liver sections by a modified two-step perfusion technique. Isolated cells were cryopreserved and stored in liquid nitrogen for one to twelve months. The total yield of freshly isolated hepatocytes averaged 3.7×10⁷ cells per gram of wet tissue. Based on trypan blue exclusion, fresh preparations contained an average of 85% viable hepatocytes vs. 70% in cryopreserved samples. The plating efficiencies of cells seeded immediately after isolation ranged from 87% to 98%, while those of cryopreserved/thawed cells were markedly lower. Flow cytometry analysis of cells labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) showed that there was no significant difference in viability compared with trypan blue staining. Both freshly isolated hepatocytes and those recovered from cryopreservation showed typical and intact morphology as demonstrated by light and electron microscopy. The product of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reaction was always expressed more intensely in cultures of freshly isolated hepatocytes. Measurements of lactate dehydrogenase (LDH) leakage were inversely correlated with trypan blue exclusion and CFSE labeling. Energy status, evaluated by the intracellular ATP concentration measurements, and various liver-specific functions such as urea synthesis and metabolism of 7-ethoxycoumarin were maintained both in fresh and cryopreserved/thawed hepatocytes. However, the activities were expressed at different levels in thawed cells. These data illustrate the importance and feasibility of human hepatocyte banking. In addition, it is clear that further refinements in the methods of hepatocyte isolation and cryopreservation are needed to utilize more fully these valuable cells in the clinic.     

Human embryonic stem cell technologies and drug discovery

Development of new drugs is costly and takes huge resources into consideration. The big pharmaceutical companies are currently facing increasing developmental costs and a lower success‐rate of bringing new compounds to the market. Therefore, it is now of outmost importance that the drug‐hunting companies minimize late attritions due to sub‐optimal pharmacokinetic properties or unexpected toxicity when entering the clinical programs. To achieve this, a strong need to test new candidate drugs in assays of high human relevance in vitro as early as possible has been identified. The traditionally used cell systems are however remarkably limited in this sense, and new improved technologies are of greatest importance. The human embryonic stem cells (hESC) is one of the most powerful cell types known. They have not only the possibility to divide indefinitely; these cells can also differentiate into all mature cell types of the human body. This makes them potentially very valuable for pharmaceutical development, spanning from use as tools in early target studies, DMPK or safety assessment, as screening models to find new chemical entities modulating adult stem cell fate, or as the direct use in cell therapies. This review illustrates the use of hESC in the drug discovery process, today, as well as in a future perspective. This will specifically be exemplified with the most important cell type for pharmaceutical development—the hepatocyte. We discuss how hESC‐derived hepatocyte‐like cells could improve this process, and how these cells should be cultured if optimized functionality and usefulness should be achieved. J. Cell. Physiol. 219: 513–519, 2009. © 2009 Wiley‐Liss, Inc.     

Cryopreservation of adult human hepatocytes obtained from resected liver biopsies

Isolated human hepatocytes have been shown to represent a valuable in vitro model to investigate the metabolism and cytotoxicity of xenobiotics. In addition, human hepatocyte transplantation and artificial liver support systems using isolated human hepatocytes are currently investigated as treatment for acute and chronic hepatic failure. In this regard, human hepatocyte banking by cryopreservation would be of great interest. In the present study, freshly isolated hepatocytes from resected liver biopsies of 28 separate donors (viability: 88 +/- 2%; plating efficiency: 79 +/- 5%) were cryopreserved using two different protocols, stepwise freezing (SF) or progressive freezing (PF), in combination (PF(+), SF(+)) or not (PF(-), SF(-)) with a 30 min preincubation in culture medium at 37 degrees C. Total recovery was higher after PF (38 +/- 3%) than after SF (12 +/- 2%). Preincubation prior to SF had no effect on plating efficiency of thawed hepatocytes (SF(-): 38 +/- 6% versus SF(+): 46 +/- 7%) while preincubation prior to PF increased plating efficiency of thawed hepatocytes (PF(-): 42 +/- 6% versus PF(+): 64 +/- 4%, p < 0.05). In attached cultured human cryopreserved/thawed hepatocytes (CH) from the PF(+) group, albumin production and glutathione content were not significantly different from those of the freshly isolated hepatocyte (FIH) cultures. Cells in CH monolayers appeared smaller than cells in FIH monolayers. In addition, the pattern of cytochrome P450- and UDP-glucuronosyl transferase-dependent isoenzyme activities and GST activity were different, suggesting a variability in the resistance to cryopreservation of the various liver hepatocyte populations. Taken all together, the results of the present study suggest that recovery of human hepatocytes after isolation prior to progressive freezing should allow human hepatocyte banking for use in pharmacotoxicology and cell therapy research purposes.     

Determining “safe” levels of exposure: The validity of the use of 10X safety factors

The current guideline for risk assessment of chemicals having a toxic end point routinely uses the reference dose (RfD) approach based on uncertainty factors of 10. With this method the quality of individual risk assessment varies among chemicals, often resulting in either an over‐ or under‐estimation of adverse health risk. The purpose of this investigation is to evaluate whether the magnitude of the 10X uncertainty factors have scientific merit against data from published experimental studies. A compilation and comparison of ratios between LOAEL/NOAEL (Lowest Observed Adverse Effect Level/No Observed Adverse Effect Level), subchronic/chronic, and animal/human values were made. The results of the present investigation revealed that the use of default factors could be over‐conservative or unprotective. More reasonable estimates of the risk to human health would result in a reduction of unnecessary, and expensive over‐regulation. In addition to the LOAEL to NOAEL, and subchronic to chronic ratios, the adequacy of uncertainty factors for animal to human extrapolations were examined. Although a 10‐fold uncertainty factor (UF) is most commonly used in the risk assessment process, an examination of the literature for the compounds presented here suggests that the use of different values is scientifically justifiable.     

Isolated hepatocytes - past, present and future

The first technique for large-scale preparation of isolated hepatocytes was described in 1953 and involved perfusion of rat liver under pressure with a Ca2+-free solution containing a chelating agent. Various modifications of this technique were in use over the next ten years, until it was demonstrated that cells prepared in this manner were grossly damaged, losing most of their cytoplasmic enzymes during the preparative procedure. The successful preparation of intact isolated hepatocytes by collagenase-treatment of liver was achieved in 1967, and the widespread use of intact hepatocyte suspensions was accelerated by the development soon after of high-yield preparative techniques involving perfusion of the liver with a medium containing collagenase.The introduction of the isolated hepatocyte preparation has enabled experimental studies that otherwise would not be feasible. Important advances have been the use of cultured hepatocytes, frequently of human origin, for the investigation of the metabolism and toxicology of potential therapeutic agents. Success in this field has been achieved through the steady improvement in techniques for the maintenance in culture of differentiated hepatocytes, and in particular their cytochrome P450 complexes. Another area showing considerable promise is the employment of hepatocytes, generally from a porcine source, in temporary support systems for patients with acute liver failure. Our own studies have concentrated on the demonstration of long-range interactions between hepatocyte compartments which suggest that energy transfer between cell compartments can take place without ATP turnover.     

Differential sensitivity of human hepatoma cell line and primary rat hepatocyte culture to benzo(a)pyrene-induced unscheduled DNA synthesis and adduct formation.

We studied the genotoxic potential of a carcinogen in the human hepatoma cell line, HepG2 and in primary rat hepatocyte culture. HepG2 is a well differentiated human hepatoblastoma cell line with biotransforming capacity. Rat hepatocytes were obtained by the standard two-step in situ perfusion technique. Following benzo(a)pyrene treatment, both HepG2 and primary rat hepatocyte culture showed unscheduled DNA synthesis with different sensitivity. In ³²P-postlabelling analysis, the chromatogram revealed quantitative and qualitative differences between HepG2 and primary rat hepatocyte cultures when treated with 10 μM benzo(a)pyrene for 18 hr. The results have demonstrated that the HepG2 cell line may be used in addition to primary rat hepatocytes in risk assessment for detection of environmental carcinogens.     

Activation of mutagens by hepatocytes and liver 9000 X g supernatant from human origin in the Salmonella typhimurium mutagenicity assay. Comparison with rat liver preparations

The mutagenicity of 10 known genotoxic compounds, of several chemical classes, was measured in Salmonella typhimurium mutagenicity assays comprising isolated human hepatocytes or human liver 9000 X g supernatant (S9) from 4 different individuals, as activating system. The mutagenic activity of several compounds as determined with the Salmonella/hepatocyte suspension assay showed obvious differences when compared with the values obtained in the Salmonella/S9 plate assay. For instance, the mutagenic activity of BZ, DMN and DEN appeared to be much higher in the hepatocyte assay than in the S9 assay. However, 2-AF and 2-AAF were activated more effectively into mutagens in the S9 assay than in the hepatocyte assay. 2-AF was slightly more mutagenic than 2-AAF in the hepatocyte assay, whereas it was far more mutagenic than 2-AAF in the S9 assay. DMN was found more mutagenic than DEN in the hepatocyte assay, whereas in the S9 assay DEN appeared to be slightly more mutagenic. Furthermore, great interindividual differences in the metabolic activation of certain compounds, e.g. BZ and DMN, were observed in the hepatocyte suspension assay, whereas these variations were less evident in the S9 plate assay. Comparison of the mutagenicity data obtained with the human liver preparations, with those obtained with rat liver preparations, showed great interspecies differences in the capacity to activate certain chemicals into mutagens. The use of human liver preparations, in particular isolated human hepatocytes, may be of great value in studies on inter- and intraspecies variations in metabolic activation of genotoxic agents.     

Assessment of cell concentration and viability of isolated hepatocytes using flow cytometry

The assessment of cell concentration and viability of freshly isolated hepatocyte preparations has been traditionally performed using manual counting with a Neubauer counting chamber and staining for trypan blue exclusion. Despite the simple and rapid nature of this assessment, concerns about the accuracy of these methods exist. Simple flow cytometry techniques which determine cell concentration and viability are available yet surprisingly have not been extensively used or validated with isolated hepatocyte preparations. We therefore investigated the use of flow cytometry using TRUCOUNT Tubes and propidium iodide staining to measure cell concentration and viability of isolated rat hepatocytes in suspension. Analysis using TRUCOUNT Tubes provided more accurate and reproducible measurement of cell concentration than manual cell counting. Hepatocyte viability, assessed using propidium iodide, correlated more closely than did trypan blue exclusion with all indicators of hepatocyte integrity and function measured (lactate dehydrogenase leakage, cytochrome p450 content, cellular ATP concentration, ammonia and lactate removal, urea and albumin synthesis). We conclude that flow cytometry techniques can be used to measure cell concentration and viability of isolated hepatocyte preparations. The techniques are simple, rapid, and more accurate than manual cell counting and trypan blue staining and the results are not affected by protein-containing media.     

Influence of Pre-, Intra- and Post-Operative Parameters of Donor Liver on the Outcome of Isolated Human Hepatocytes

The aim of the present study was to analyse, retrospectively on a large panel of patients (149), the influence of the donor liver characteristics on the outcome of human hepatocyte isolation obtained from resected liver biopsies from surgical waste after hepatectomy. Among the pre-operative parameters, the type of disease, age and sex of the patient, previous chemotherapy, alcohol or tobacco consumption did not affect the yield, viability, attachment rate and function of the isolated human hepatocytes. Pre-operative biological and anatomopathological data indicated that, while mild steatosis (≤10% steatotic hepatocytes) did also not affect the outcome of hepatocyte isolation, stronger steatosis (>10% steatotic hepatocytes) tended to decrease hepatocyte yield. Cholestasis, as assessed by γ-glutamyl transferase serum values, significantly negatively correlated with the percentage of digested liver and the yield of viable cells. Intra-operative clamping time, that is, warm ischaemia, longer than 30 min was found to decrease both the percentage of digested liver and cell yield. Among the post-operative parameters, the percentage of digested liver decreased when biopsy weights were higher than 100 g, the use of glue tended to increase both the percentage of digested tissue and the yield of viable cells. In conclusion, human diseased livers appear to be a valuable source of isolated functional human hepatocytes. We recommend, for an optimal isolation, to use liver biopsies weighing less than 100 g, to glue the section surfaces of the biopsies and to avoid the use of moderate steatotic livers (>10% steatotic hepatocytes) and cholestatic livers, as well as livers undergoing warm ischaemia or clamping during resection due to the decrease in cell yield. This revised version was published online in July 2006 with corrections to the Cover Date.     

Clonal mesenchymal stem cells derived from human bone marrow can differentiate into hepatocyte‐like cells in injured livers of SCID mice

There is increasing evidence that human mesenchymal stem cells (hMSCs) can be a valuable, transplantable source of hepatocytes. Most of the hMSCs preparations used in these studies were likely heterogeneous cell populations, isolated by adherence to plastic surfaces or by density gradient centrifugation. Therefore, the participation of other unknown trace cell populations cannot be rigorously discounted. Here we report the isolation and establishment of a cloned human MSC line (chMSC) from human bone marrow primary culture, through which we confirmed the hepatic differentiation capability of authentic hMSCs. chMSCs expressed markers of mesenchymal cells, but not markers of hematopoietic stem cells. In vitro, chMSCs can differentiate into either mesenchymal cells or cells exhibiting hepatocyte‐like phenotypes. When transplanted intrasplentically into carbon tetrachloride‐injured livers of SCID mice, EGFP‐tagged chMSCs engrafted into the host liver parenchyma, exhibited typical hepatocyte morphology, form a three‐dimensional architecture, and differentiate into hepatocyte‐like cells expressing human albumin and α‐1‐anti‐trypsin. By confocal microscopy, ultrafine intercellular nanotubular structures were visible between adjacent transplanted and host hepatocytes. We postulate that these structures may assist in the phenotype conversion of chMSCs, possibly by exchange of cytoplasmic components between native hepatocytes and transplanted cells. Thus, a clonal pure population of hMSCs, which can be expanded in culture, may have potential as a cellular source for substitution damaged cells in hepatic injury. J. Cell. Biochem. 108: 693–704, 2009. © 2009 Wiley‐Liss, Inc.     

The Cumulative Risk to Human Health of Pharmaceuticals in New Jersey Surface Water

Surface water in New Jersey is used by many residential drinking water facilities. Like many water sources it is contaminated by upstream industrial and residential sources, including pharmaceutical residues. This research examines the concentrations of 18 pharmaceuticals in 30 New Jersey locations, their acceptable daily exposures (ADE), and potential drug–drug interactions (DDI). The surface water data was provided by the U.S. Geological Survey (USGS). ADEs for human health were set for each pharmaceutical in the study. The pharmaceuticals were evaluated for known adverse health interactions and their potential health impact. These factors were brought together using a cumulative hazard index (HI) risk assessment calculation to assess the overall risk of pharmaceuticals in NJ surface water to human health. When examining the potential for DDI in this assessment, the risk increased but not appreciably. The HI for the sample locations ranged from <0.00001 to 0.01 with the DDI adding less than 1.2× increase to the overall risk. The calculated risk of these mixtures was also increased to an extreme DDI of 7 times per interaction. A noticeable increase in the calculated risk was seen, but in no cases did it reach a level of concern.     

Isolation and primary culture of adult human hepatocytes

Summary Biopsy tissue of adult human liver was gently dissociated with collagenase followed by Dispase. By repeated low g centrifugation, a large number of almost pure, viable hepatocytes was obtained. This is the first report of a successful procedure for obtaining adult human hepatocytes for study in tissue culture. The isolated cells have the typical morphology of liver parenchyma, and these characteristics persist throughout the period of culturing. Evidence of their function is indicated by albumin synthesis. This procedure is now being used to study human hepatocyte functions in vitro and the effects of a variety of agents including carcinogens and viruses.     

Human liver model systems in a dish

The human adult liver has a multi‐cellular structure consisting of large lobes subdivided into lobules containing portal triads and hepatic cords lined by specialized blood vessels. Vital hepatic functions include filtering blood, metabolizing drugs, and production of bile and blood plasma proteins like albumin, among many other functions, which are generally dependent on the location or zone in which the hepatocyte resides in the liver. Due to the liver's intricate structure, there are many challenges to design differentiation protocols to generate more mature functional hepatocytes from human stem cells and maintain the long‐term viability and functionality of primary hepatocytes. To this end, recent advancements in three‐dimensional (3D) stem cell culture have accelerated the generation of a human miniature liver system, also known as liver organoids, with polarized epithelial cells, supportive cell types and extra‐cellular matrix deposition by translating knowledge gained in studies of animal organogenesis and regeneration. To facilitate the efforts to study human development and disease using in vitro hepatic models, a thorough understanding of state‐of‐art protocols and underlying rationales is essential. Here, we review rapidly evolving 3D liver models, mainly focusing on organoid models differentiated from human cells.     

Use of Percoll density gradient centrifugation for preparing isolated rat hepatocytes having long-term viability

A method for preparing suspensions of hepatocytes with long-term viability is described. Its originality lies in the use, after conventional preparation, of a Percoll density gradient centrifugation which allows a complete and rapid separation of cell debris and released proteases from the intact hepatocytes. A series of functional reactions characterizing drug metabolism (P-450 level, ethoxycoumarin hydroxylation, methylumbelliferone conjugation) and membrane transport and integrity (α-aminoisobutyric acid transport and its stimulation by both insulin and glucagon) were conducted, in parallel to the trypan blue exclusion test, to evaluate the metabolic activity of both conventional and Percoll preparations. This investigation clearly demonstrates that the use of Percoll density gradient centrifugation significantly increases (from 5–6 to 30–35 h) the half-life of freshly isolated hepatocyte suspensions.     

沈阳某冶炼厂废弃厂区的人类健康风险评价

以沈阳某冶炼厂废弃厂区重金属污染监测为依据,采用美国环保局最新的人类健康风险评价标准方法对沈阳某冶炼厂废弃地块污染土壤进行了评价,并且假设未来该土地利用类型为工业用地(Ⅰ)或休闲用地(Ⅱ).评价结果显示:工业用地(Ⅰ)和休闲用地(Ⅱ)的累积非致癌风险指数分别为2.65×10-2和3.67×10-2;工业用地(Ⅰ)和休闲用地(Ⅱ)由呼吸摄入Cd造成的潜在致癌风险指数分别为4.48×10-9和7.30×10-10,不会对在该地区工作和休闲的人们造成身体健康上的伤害;无论是工业用地假设还是休闲用地假设,由无机铜造成的人类健康风险在整个风险中所占的比例最大;由美国环保局的人类健康风险评价方法反推得出的冶炼厂地块未来为工业用地的土壤修复目标值均小于我国工业企业土壤环境质量风险评价基准值.