Regulatory landscape of alternatives to animal testing in food safety evaluations with a focus on the western world.

Traditional animal models are increasingly being replaced by new approach methodologies (NAMs) which focus on predicting toxicity of chemicals based on mechanistic data rather than apical endpoint data usually obtained from animal models. Beyond in vitro genetic tests, however, only a handful of NAMs have been successfully implemented in regulatory decision-making processes, mostly in the cosmetics and chemicals sector. Regulatory guidance on food safety testing in many jurisdictions still relies on data obtained from animal studies. This is due to the lack of validated models to predict systemic toxicity, which is essential to develop health-based guidance values for food additives. Other factors limiting the adoption of NAMs into food safety assessment include sector legislation lagging behind scientific progress, and lack of training and expertise to use the new models. While regulatory and industry bodies are working to combat these challenges, more needs to be done before these models can be used as standalone tools for regulatory decision-making. This review summarizes the current state and challenges of regulatory acceptance of NAMs for decision-making, and the efforts by governing bodies and industry to transition from animal testing for food safety assessments.

Comparative In vitro metabolism of purified mogrosides derived from monk fruit extracts.

Mogrosides are the primary components responsible for the sweet taste of Monk fruit which is derived from Siraitia grosvenorii (Swingle), a herbaceous plant native to southern China. Many mogrosides have been identified from Monk fruit extract, but the major sweetness component of Monk fruit by mass is mogroside V, comprising up to 0.5% of the dried fruit weight. Recent pharmacokinetic studies indicate that the parent mogrosides undergo minimal systemic absorption following ingestion and hydrolysis by digestive enzymes and/or intestinal flora and are excreted as mogrol (i.e., the aglycone) and its mono- and diglucosides. The objective of this study was to demonstrate whether individual mogrosides, are metabolized to a common and terminal deglycosylated metabolite, mogrol. An in vitro assay was conducted with pooled human male and female intestinal fecal homogenates (HFH) using mogrosides IIIe, mogroside V, siamenoside I, and isomogroside V at two concentrations over a 48 h period. The results show that various mogrosides that differ in the linkages and number of glucose units attached to a common cucurbitane backbone, share a common metabolic fate, and are metabolized within 24 h to mogrol. Aside from an apparent difference in the initial rate of deglycosylation between mogrosides at higher concentrations, no apparent difference in the rate of deglycosylation was observed between the male and female HFH. Given the similar structures of these mogrosides and a shared metabolic fate to mogrol, the study provides support for a reasonably conservative approach to assess safety based on bridging safety data from an individual mogroside (i.e., Mogroside V) to other mogrosides, and the establishment of a group Acceptable Daily Intake (ADI), rather than individual ADI's for mogrosides.

Alternatives to animal testing in toxicity testing: Current status and future perspectives in food safety assessments.

The development of alternative methods to animal testing has gained great momentum since Russel and Burch introduced the "3Rs" concept of Reduction, Refinement, and Replacement of animals in safety testing in 1959. Several alternatives to animal testing have since been introduced, including but not limited to in vitro and in chemico test systems, in silico models, and computational models (e.g., [quantitative] structural activity relationship models, high-throughput screens, organ-on-chip models, and genomics or bioinformatics) to predict chemical toxicity. Furthermore, several agencies have developed robust integrated testing strategies to determine chemical toxicity. The cosmetics sector is pioneering the adoption of alternative methodologies for safety evaluations, and other sectors are aiming to completely abandon animal testing by 2035. However, beyond the use of in vitro genetic testing, agencies regulating the food industry have been slow to implement alternative methodologies into safety evaluations compared with other sectors; setting health-based guidance values for food ingredients requires data from systemic toxicity, and to date, no standalone validated alternative models to assess systemic toxicity exist. The abovementioned models show promise for assessing systemic toxicity with further research. In this paper, we review the current alternatives and their applicability and limitations in food safety evaluations.

Water adherence factors for human skin.

On incidental dermal exposure to chemicals in water, a key exposure factor is the amount of water adhering to skin. Although soil adherence factors have been developed for risk assessment, measurements of water adherence on human skin have not been described. In the Environmental Protection Agency's (EPA's) dermal risk assessment guidance, dermal dose from environmental exposures is based upon the flux rate across the skin, which assumes that an unlimited amount of chemical is available for absorption. This assumption is applicable to certain exposure scenarios such as swimming and bathing. However, exposures to contaminated water frequently involve scenarios where the available chemical is limited by the amount of water adhering to the skin, for example, during accidental splashes.

Chlorotyrosine protein adducts are reliable biomarkers of neutrophil-induced cytotoxicity in vivo.

INTRODUCTION: A limitation for investigating the pathophysiological role of neutrophils in vivo is the lack of a reliable biomarker for neutrophil cytotoxicity in the liver. Therefore, we investigated if immunohistochemical detection of chlorotyrosine protein adducts can be used as a specific footprint for generation of neutrophil-derived hypochlorous acid in vivo. METHODS: C3Heb/FeJ mice were treated with 100 micrograms/kg endotoxin (ET) alone or in combination with 700 mg/kg galactosamine (Gal/ET). Some animals received additionally two doses of 10 mg/kg of the pancaspase inhibitor Z-VAD-fmk. An antibody against chlorotyrosine was used for the immunohistochemical analysis. RESULTS: At 6 h after Gal/ET, hepatocellular apoptosis was evident without increase in plasma ALT activities. Neutrophils accumulated in sinusoids but there was no evidence for chlorotyrosine staining. At 7 h after Gal/ET, about 54% of the sequestered neutrophils had extravasated, there was extensive necrosis and increased plasma ALT activities. Extensive immunostaining for chlorotyrosine, mainly colocalized with neutrophils, could be observed. Treatment with Z-VAD-fmk eliminated apoptosis, necrosis and the increase in plasma ALT values. Neutrophil extravasation was prevented but the overall number of neutrophils in the liver was unchanged. Chlorotyrosine staining was absent in these samples. After ET alone (7 h), sinusoidal neutrophil accumulation was similar to Gal/ET treatment but there was no apoptosis, neutrophil extravasation, ALT release or chlorotyrosine staining. CONCLUSIONS: Chlorotyrosine staining in liver samples correlated well with evidence of neutrophil-induced liver injury in the endotoxemia model. These results indicate that assessment of chlorotyrosine protein adduct formation by immunohistochemistry could be a useful marker of neutrophil-induced liver cell injury in vivo.

Mode of cell death after acetaminophen overdose in mice: apoptosis or oncotic necrosis?

Acetaminophen (AAP) overdose can cause severe liver injury and liver failure in experimental animals and humans. Recently, several authors proposed that apoptosis might be a major mechanism of cell death after AAP treatment. To address this controversial issue, we evaluated a detailed time course of liver injury after AAP (300 mg/kg) in fasted C3Heb/FeJ mice. Apoptotic hepatocytes were quantified in H&E-stained liver sections using morphologic criteria (cell shrinkage, chromatin condensation and margination, and apoptotic bodies). The number of apoptotic hepatocytes remained at baseline (0.2 +/- 0.1 cells/10 high-power fields [HPF]) up to 2 h after AAP administration. However, between 3 and 24 h, apoptotic cell death increased significantly, e.g., 6.3 +/- 0.8 cells/10 HPF at 6 h. Despite the increase in the number of hepatocytes meeting the morphological criteria of apoptosis, this cell fraction remained well below 1% of all parenchymal cells. No evidence for caspase-3 processing or increase in enzyme activity was detected at any time. These results were compared to the overall percent of necrotic cells in liver sections. Confluent areas of centrilobular necrosis were estimated to involve 40-60% of all hepatocytes between 3 and 24 h after AAP administration. These numbers correlated with the increase in plasma alanine aminotransferase activities, which reached a peak level of 5900 +/- 1350 U/l at 24 h. A similar result was obtained with higher doses of AAP and with the use of fed animals. Thus, oncotic necrosis and not apoptosis is the principal mechanism of liver-cell death after AAP overdose in vivo.

Hepatobiliary transporter expression in intercellular adhesion molecule 1 knockout and Fas receptor-deficient mice after common bile duct ligation is independent of the degree of inflammation and oxidative stress.

Liver injury in intercellular adhesion molecule 1 knockout (ICAM(-/-)) and Fas receptor-deficient (lpr) mice is markedly reduced after common bile duct ligation (CBDL) due to significantly reduced inflammation and oxidative stress. Liver injury in CBDL rodents is counteracted by adaptive hepatobiliary transporter induction. Since hepatobiliary transporter expression in obstructive cholestasis may be regulated not only by accumulating bile acids but also by inflammatory mediators and oxidative stress, we hypothesized that differences in the inflammatory response may affect hepatobiliary transporter expression in CBDL, which would contribute to reduced liver injury. Therefore, expression of major hepatobiliary transporters (Ntcp, Bsep, Mrp2-4, Ost alpha/beta) was determined by Taqman RT-PCR and Western blotting in sham-operated animals and 3 days after CBDL in wild-type, ICAM(-/-) and lpr mice of the endotoxin-sensitive C57BL/6 and the endotoxin-resistant C3H/HeJ strains. CBDL resulted in a significant decrease of Ntcp in all genotypes. Canalicular transporters Bsep and Mrp2 were repressed only in the endotoxin-sensitive strain regardless of the genotype. Mrp3 was moderately induced in ICAM(-/-), lpr, and endotoxin-resistant mice, whereas Mrp4 was only induced in the endotoxin-resistant strain. Ost beta was massively induced in all CBDL mice, whereas Ost alpha was reduced. In conclusion, markedly reduced inflammation and oxidative stress in CBDL ICAM(-/-) and lpr mice does not profoundly affect hepatobiliary transporter expression. Therefore, transporter expression does not account for reduced liver injury in ICAM(-/-) and lpr mice. Induction of the adaptive transporter response after CBDL is independent of the degree of the inflammatory response. Rather, retention of biliary constituents may determine transporter expression in CBDL.

Generation and functional significance of CXC chemokines for neutrophil-induced liver injury during endotoxemia.

The hypothesis that the neutrophil chemoattractant CXC chemokines KC and macrophage inflammatory protein-2 (MIP-2) are involved in neutrophil transmigration and liver injury was tested in C3Heb/FeJ mice treated with galactosamine (Gal, 700 mg/kg), endotoxin (ET, 100 microg/kg), or Gal + ET (Gal/ET). Hepatic KC and MIP-2 mRNA levels and plasma CXC chemokine concentrations were dramatically increased 1.5 h after Gal/ET or ET alone and gradually declined up to 7 h. Murine recombinant cytokines (TNF-alpha, IL-1 alpha, and IL-1 beta), but not Gal/ET, induced CXC chemokine formation in the ET-resistant C3H/HeJ strain. To assess the functional importance of KC and MIP-2, C3Heb/FeJ mice were treated with Gal/ET and control IgG or a combination of anti-KC and anti-MIP-2 antibodies. Anti-CXC chemokine antibodies did not attenuate hepatocellular apoptosis, sinusoidal neutrophil sequestration and extravasation, or liver injury at 7 h. Furthermore, there was no difference in liver injury between BALB/cJ wild-type and CXC receptor-2 gene knockout (CXCR2-/-) mice treated with Gal/ET. The higher neutrophil count in livers of CXCR2-/- than in wild-type mice after Gal/ET was caused by the elevated number of neutrophils located in sinusoids of untreated CXCR2-/- animals. The pancaspase inhibitor Z-Val-Ala-Asp-fluoromethylketone eliminated Gal/ET-induced apoptosis and neutrophil extravasation and injury but not CXC chemokine formation. Thus Gal/ET induced massive, cytokine-dependent CXC chemokine formation in the liver. However, neutrophil extravasation and injury occurred in response to apoptotic cell injury at 6-7 h and was independent of CXC chemokine formation.

Apoptosis and necrosis in liver disease.

Liver cell injury and cell death is a prominent feature in all liver disease processes. During the last 5-10 years, most research activities focused almost exclusively on evaluating apoptotic cell death and the corresponding intracellular signaling pathways. Although this effort led to substantial progress in our understanding of the mechanisms of apoptosis, it also created substantial confusion regarding the predominant mode of cell death and the relevance of apoptosis in a variety of liver disease models, as discussed in this review for acetaminophen and troglitazone hepatotoxicity, obstructive cholestasis and viral hepatitis. Part of the problem is related to the fact that there is no specific assay or parameter, with the exception of morphological changes in vivo, which allows the unequivocal distinction between apoptosis and oncotic necrosis. In addition, some aspects of the signaling pathways are similar. Therefore, to make progress in identifying relevant pharmacological intervention strategies to prevent or attenuate human liver disease processes, it is of critical importance to apply several different experimental approaches and analyze as many parameters as possible. In addition, positive controls for the assumed process should be used whenever possible and mechanisms of cell injury should only be investigated in model systems relevant for the human pathophysiology.

Reduced oncotic necrosis in Fas receptor-deficient C57BL/6J-lpr mice after bile duct ligation.

Neutrophils aggravate cholestatic liver injury after bile duct ligation (BDL). Recently, it was suggested that hepatocellular apoptosis might be critical for liver injury in this model. To test the hypothesis that apoptosis could be a signal for neutrophil extravasation and injury, we assessed parameters of apoptosis and inflammation after BDL using 2 different approaches: (1) wild-type and Fas receptor-deficient lpr mice of the C57BL/6J or C3H/HeJ strains, and (2) treatment with the pancaspase inhibitor z-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk)in C3HeB/FeJ mice. After BDL for 3 days, total cell death was estimated to be between 10% and 50% of all cells evaluated. However, less than 0.1% of hepatocytes showed apoptotic morphology in all 3 strains. Processing of procaspase-3, caspase-3 enzyme activities, and immunohistochemical staining for cytokeratin 18 cleavage products indicated no activation of caspases. Real-time reverse-transcriptase polymerase chain reaction analysis revealed increased expression of many inflammatory mediators but no effect on proapoptotic genes. More than 50% of all accumulated neutrophils were extravasated and colocalized with foci of oncotic hepatocytes and chlorotyrosine adducts. z-VAD-fmk treatment had no effect on apoptosis or liver injury after BDL but eliminated apoptosis after galactosamine/endotoxin in C3HeB/FeJ mice. In Fas receptor-deficient lpr mice (C57BL/6J), expression of inflammatory mediators, neutrophil accumulation and extravasation, chlorotyrosine adduct formation, and liver injury were reduced. This protection was not observed in lpr mice of the endotoxin-resistant C3H/HeJ strain. In conclusion, liver injury (oncotic necrosis) after BDL correlated with the severity of the inflammatory response. The minimal amount of apoptosis had no effect on inflammation or on the overall injury.

Oncotic necrosis and caspase-dependent apoptosis during galactosamine-induced liver injury in rats.

The mode of cell death during galactosamine (Gal)-induced liver injury was originally thought to be oncotic necrosis but recently it was suggested to be apoptosis. Thus, the objective was to assess whether apoptosis and oncosis are sequential or independent events in the pathophysiology. In addition, the role of caspases in Gal-induced apoptotic signaling was investigated. A dose of 500 mg/kg Gal caused a time-dependent increase in plasma alanine transaminase (ALT) levels (24 h: 430 +/- 122 U/L) in female Sprague-Dawley rats. This was accompanied by processing of procaspase-3 and significant increases in hepatic and plasma caspase-3 activities. Using morphology and TUNEL staining, apoptotic and oncotic cells were quantitated. The number of apoptotic hepatocytes increased from 0.14% in controls to 5.4 +/- 1.0% 24 h after Gal treatment. In addition, the number of cells with oncotic morphology increased from 0 to 6.9% of total hepatocytes. Treatment with the pan-caspase inhibitor IDN-7314 (10 mg/kg) or pretreatment with uridine (1 g/kg), reduced all parameters of apoptosis to baseline. However, IDN-7314 administration did not affect plasma ALT activities and the number of oncotic cells at 6 h and only modestly reduced these parameters at 24 h. Uridine, on the other hand, prevented the increase of plasma ALT levels and reduced the number of apoptotic and oncotic cells by >80%. In conclusion, galactosamine-induced hepatocellular apoptosis in rats is caspase dependent. Although some of the apoptotic cells may undergo secondary necrosis, a significant number of hepatocytes die through oncotic necrosis as an independent mechanism of cell death.

NADPH oxidase-derived oxidant stress is critical for neutrophil cytotoxicity during endotoxemia.

Neutrophils can cause liver injury during endotoxemia through generation of reactive oxygen species. However, the enzymatic source of the oxidant stress and the nature of the oxidants generated remain unclear. Therefore, we investigated the involvement of NADPH oxidase in the pathophysiology by using the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) in the galactosamine/endotoxin (700 mg/kg Gal:100 microg/kg ET) model of liver injury. In addition, we measured chlorotyrosine as indicator for hypochlorous acid formation by myeloperoxidase. Gal/ET treatment of male C3HeB/FeJ mice resulted in sinusoidal neutrophil accumulation and parenchymal cell apoptosis (14 +/- 3% of cells) at 6 h. At 7 h, 35% of neutrophils had transmigrated. The number of apoptotic cells increased to 25 +/- 2%, and the overall number of dead cells was 48 +/- 3%; many of them showed the characteristic morphology of necrosis. Hepatocytes, which colocalized with extravasated neutrophils, stained positive for chlorotyrosine and 4-hydroxynonenal (4-HNE) protein adducts. In contrast, animals pretreated with DPI (2.5 mg/kg) were protected against liver injury at 7 h (necrosis = 20 +/- 2%). These livers showed little chlorotyrosine or 4-HNE staining, but apoptosis and neutrophil accumulation and extravasation remained unaffected. However, DPI-treated animals showed serious liver injury at 9 h due to sustained apoptosis. The results indicate that NADPH oxidase is responsible for the neutrophil-derived oxidant stress, which includes formation of hypochlorous acid by myeloperoxidase. Thus NADPH oxidase could be a promising therapeutic target to prevent neutrophil-mediated liver injury. However, the long-term benefit of this approach needs to be investigated in models relevant for human liver disease.

Neutrophils aggravate acute liver injury during obstructive cholestasis in bile duct-ligated mice.

Obstruction of the common bile duct in a variety of clinical settings leads to cholestatic liver injury. An important aspect of this injury is hepatic inflammation, with neutrophils as the prominent cell type involved. However, the pathophysiologic role of the infiltrating neutrophils during cholestatic liver injury remains unclear. Therefore, we tested the hypothesis that neutrophils contribute to the overall pathophysiology by using bile duct-ligated (BDL) wild-type animals and mice deficient in the beta(2) integrin CD18. In wild-type animals, neutrophils were activated systemically as indicated by the increased expression of Mac-1 (CD11b/CD18) and L-selectin shedding 3 days after BDL. Histologic evaluation (48 +/- 10% necrosis) and plasma transaminase levels showed severe liver injury. Compared with sham-operated controls (< 10 neutrophils per 20 high-power fields), large numbers of neutrophils were present in livers of BDL mice (425 +/- 64). About 60% of these neutrophils had extravasated into the parenchyma. In addition, a substantial number of extravasated neutrophils were found in the portal tract. In contrast, Mac-1 was not up-regulated and plasma transaminase activities and the area of necrosis (21 +/- 9%) were significantly reduced in CD18-deficient animals. These mice had overall 62% less neutrophils in the liver. In particular, extravasation from sinusoids and portal venules (PV) was reduced by 91% and 47%, respectively. Immunohistochemical staining for chlorotyrosine, a marker of neutrophil-derived oxidant stress, was observed in the parenchyma of BDL wild-type but not CD18-deficient mice. In conclusion, neutrophils aggravated acute cholestatic liver injury after BDL. This inflammatory injury involves CD18-dependent extravasation of neutrophils from sinusoids and reactive oxygen formation.

Functional importance of ICAM-1 in the mechanism of neutrophil-induced liver injury in bile duct-ligated mice.

Cholestasis-induced liver injury during bile duct obstruction causes an acute inflammatory response. To further characterize the mechanisms underlying the neutrophil-induced cell damage in the bile duct ligation (BDL) model, we performed experiments using wild-type (WT) and ICAM-1-deficient mice. After BDL for 3 days, increased ICAM-1 expression was observed along sinusoids, along portal veins, and on hepatocytes in livers of WT animals. Neutrophils accumulated in sinusoids [358 +/- 44 neutrophils/20 high-power fields (HPF)] and >50% extravasated into the parenchymal tissue. Plasma alanine transaminase (ALT) levels increased by 23-fold, and severe liver cell necrosis (47 +/- 11% of total cells) was observed. Chlorotyrosine-protein adducts (a marker for neutrophil-derived hypochlorous acid) and 4-hydroxynonenal adducts (a lipid peroxidation product) were detected in these livers. Neutrophils also accumulated in the portal venules and extravasated into the portal tracts. However, no evidence for chlorotyrosine or 4-hydroxynonenal protein adducts was detected in portal tracts. ICAM-1-deficient mice showed 67% reduction in plasma ALT levels and 83% reduction in necrosis after BDL compared with WT animals. The total number of neutrophils in the liver was reduced (126 +/- 25/20 HPF), and 85% of these leukocytes remained in sinusoids. Moreover, these livers showed minimal staining for chlorotyrosine and 4-hydroxynonenal adducts, indicating a substantially reduced oxidant stress and a diminished cytokine response. Thus neutrophils relevant for the aggravation of acute cholestatic liver injury in BDL mice accumulate in hepatic sinusoids, extravasate into the tissue dependent on ICAM-1, and cause cell damage involving reactive oxygen formation.