Toxicological evaluation of Phospholipase D from Kitasatospora paracochleata.

Phospholipases are used extensively in the production of food ingredients, typically as processing aids, to enzymatically convert glycerophospholipids and provide functional properties in meat products or baking confections. The current study examined the safety of Phospholipase D derived from Kitasatospora paracochleata (strain No. 362-PLD) for use as a processing aid in various food applications, where it may be present in the finished products at trace levels. The safety assessment of Phospholipase D included two in vitro genotoxicity studies and a 90-day subchronic toxicity study in rats. No evidence of genotoxicity was observed in a bacterial reverse mutation test or in a chromosome aberration test. In the subchronic toxicity study, no test article-related adverse effects were observed upon Phospholipase D administration to rats at doses levels of 0, 750, 1500, and 3000 mg/kg body weight/day throughout a 90-day study period. Thus, the no-observed-adverse-effect level (NOAEL) was considered to be 3000 mg/kg body weight/day. This safety assessment supports the safe use of Phospholipase D as a processing aid in food production and the presence of trace levels in finished products.

Lactobacillus rhamnosus MP108: Toxicological evaluation.

A lyophilized powder of Lactobacillus rhamnosus MP108, a strain isolated from infant feces, has been developed for potential as a probiotic. To assess safety, three genetic toxicity studies (Ames, in vivo mouse micronucleus, and in vivo mouse spermatocyte chromosome aberration assays) and one 90-day oral rat toxicity study were conducted. Lactobacillus rhamnosus MP108 had no activity in the Ames assay, with or without metabolic activation, up to the highest dose tested (5000 µg/plate). Also, no evidence of genotoxicity was observed in the two in vivo genotoxicity assays up to the maximum dose tested of 5.6 g/kg body weight. Feeding of L. rhamnosus MP108 to SD rats at doses of 0, 0.25, 0.5, or 1.5 g/kg body weight/day had no statistically or biologically significant effects on any of the parameters measured. An evaluation of Lactobacillus spp. generally, and L. rhamnosus specifically, showed no indication of concern for translocation or pathogenicity. The results of the toxicology studies showed no indications for safety concerns. PRACTICAL APPLICATION: Lactobacillus rhamnosus MP108 is a bacterium that shows potential as a probiotic. While this bacterium has been used in food and food production, a series of studies were conducted to provide further assurance that this particular strain of L. rhamnosus is safe.

Determinants of Sweetness Preference: A Scoping Review of Human Studies.

Factors associated with sweetness preference are multi-faceted and incredibly complex. A scoping review was undertaken to identify determinants of sweetness preference in humans. Using an online search tool, ProQuest ™, a total of 99 publications were identified and subsequently grouped into the following categories of determinants: Age, dietary factors, reproductive hormonal factors, body weight status, heritable, weight loss, sound, personality, ethnicity and lifestyle, previous exposure, disease, and 'other' determinants. Methodologies amongst studies were heterogenous in nature (e.g., there was variability across studies in the sweetness concentrations tested, the number of different sweetness concentrations used to assess sweetness preference, and the methods utilized to measure sweetness preference), rendering interpretation of overall findings challenging; however, for certain determinants, the evidence appeared to support predictive capacity of greater sweetness preference, such as age during certain life-stages (i.e., young and old), being in a hungry versus satiated state, and heritable factors (e.g., similar sweetness preferences amongst family members). Recommendations for the design of future studies on sweetness preference determinants are provided herein, including an "investigator checklist" of criteria to consider.

Preclinical safety evaluation of triacylglycerol lipase QLM from Burkholderia ubonensis.

Triacylglycerol lipases are well characterized enzymes that catalyze the hydrolysis of fats. They are biotechnologically relevant enzymes and are used in a wide range of practical applications in industry. Lipase produced from Burkholderia ubonensis (strain PL266-QLM) (Lipase QLM) is being investigated for use as a processing aid in multiple food applications and may therefore be present at trace levels in finished food products. A battery of toxicological studies was therefore conducted on Lipase QLM to support its safe use in food. Lipase QLM was not genotoxic or mutagenic in an in vitro bacterial reverse mutation test and chromosome aberration test. Additionally, no test article-related adverse effects were observed in clinical observations, body weight, food consumption, ophthalmology, hematology, blood chemistry, urinalysis, and macroscopic and microscopic findings in a subchronic toxicity study in rats administered Lipase QLM in the diet at levels of 0%, 0.8%, 2%, and 5% Lipase QLM. The no-observed-adverse-effect level was therefore considered to be 5% Lipase QLM in both sexes [3357.2 and 3777.6 mg/kg body weight/day (2756.3 and 3101.4 mg total organic solids/kg body weight/day) in males and females, respectively] under the test conditions, which supports the safety of this ingredient for use in food for human consumption.

Involvement of CCL2/CCR2 macrophage recruitment in amodiaquine-induced liver injury.

Evidence suggests that macrophages may play a role in the development of idiosyncratic drug-induced liver injury (IDILI). However, there has yet to be a clear link between macrophage activation and the inflammatory infiltrate that is characteristic of IDILI. A major chemokine involved in the recruitment of macrophages into the liver is C-C motif chemokine ligand 2 (CCL2)/monocyte chemoattractant protein 1 (MCP1). Therefore, we tested the effect of this chemokine in an animal model of IDILI. Specifically, amodiaquine (AQ), which is known to cause IDILI in humans, causes mild liver injury in wild-type C57BL/6 mice that resolves despite continued AQ treatment, but it causes more severe liver injury that does not resolve in PD-1-/- mice co-treated with anti-CTLA-4 to impair immune tolerance. CCR2-/- mice treated with AQ were not protected from the expected AQ-induced liver injury seen in wild-type C57BL/6 mice. In contrast, anti-CCL2 antibodies attenuated the liver injury caused by AQ in the impaired immune tolerance model. The difference in response of the two models is likely due to a difference in the IDILI mechanism; the mild injury in wild-type animals is mediated by NK cells, while the more serious injury in the impaired immune tolerance model requires CD8 T-cells. The results from these experiments provide evidence that macrophage infiltration into the liver may not be involved in mild IDILI mediated by the innate immune system, but it does appear necessary in more severe IDILI involving cytotoxic T-cells.

The Effects of Immune Modulators on Amodiaquine-Induced Liver Injury.

If idiosyncratic drug-induced liver injury (IDILI) is immune mediated, then it is logical that immune modulators may be able to affect liver injury caused by a drug. We have previously shown that modulating the immune system by impairing programmed cell death protein (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) signaling, both receptors involved in immune tolerance, was capable of producing an animal model of amodiaquine (AQ) IDILI with characteristics very similar to IDILI in humans. Other immune modulators may also increase liver injury caused by drugs that cause IDILI in humans. In this study, myeloid derived suppressor cells (MDSCs), transforming growth factor beta (TGF-ß), and lymphocyte-activation gene 3 (LAG3) were targeted with antibodies, with and without PD-1 and CTLA-4 impairment. We found that anti-Gr1 antibodies used to deplete MDSCs led to a significant increase in AQ-induced liver injury in wild-type mice; however, the injury was actually less in PD-1-/- mice, with or without anti-CTLA-4, and it was less than we have previously observed in PD-1-/- mice combined with anti-CTLA-4 without anti-Gr1. Addition of anti-LAG3 or anti-TGF-ß antibodies produced a small increase ALT in AQ-treated wild-type mice. There was a significant increase in ALT in PD-1-/- mice co-treated with anti-LAG3 or anti-TGF-ß relative to AQ-treated wild-type mice. In the case of TGF-ß, this was further increased by the addition of anti-CTLA-4, but if anything, there appeared to be a paradoxical decrease when anti-CTLA-4 was combined with anti-LAG3. Overall, the results from this study were not always as expected, and they highlight the complexity of the immune response, in particular immune tolerance, which appears to be the dominant immune response to drugs that cause IDILI.

Use of an animal model to test whether non-alcoholic fatty liver disease increases the risk of idiosyncratic drug-induced liver injury.

Clinical evidence suggests that most idiosyncratic drug-induced liver injury (IDILI) is immune-mediated. The danger hypothesis suggests that liver injury and inflammation would increase the risk of an immune response leading to IDILI. Therefore, a reasonable hypothesis would be that an underlying chronic liver disease such as non-alcoholic steatohepatitis (NASH) would increase the risk of developing IDILI due to inflammation and release of danger signals from damaged cells. In order to test this hypothesis, mice were fed a methionine-/choline-deficient (MCD) diet that produces a consistent NASH phenotype, along with amodiaquine (AQ) - a drug known to cause IDILI in humans. This study employed both wild-type C57BL/6 mice and PD-1-/- mice co-treated with anti-CTLA-4 antibodies. The PD-1-/- + anti-CTLA-4 model produces an immune-mediated liver injury very similar to the idiosyncratic liver injury observed in humans. The liver injury observed in the present experiment was dominated by the injury caused by the MCD diet; there was no significant difference between mice treated with the MCD diet alone and those also treated with AQ, whether in wild-type mice of the PD-1-/- model. Therefore, the MCD diet, which results in a state that mimics NASH, did not appear to increase the liver injury associated with AQ treatment. Ultimately, an animal model is just that - only a model, and cannot provide a definitive answer to clinical questions. However, given the difficulty of performing clinical studies with appropriate control populations, the present results provide important evidence to support a general clinical finding that underlying liver injury does not usually increase the risk of IDILI.

Editor's Highlight: An Impaired Immune Tolerance Animal Model Distinguishes the Potential of Troglitazone/Pioglitazone and Tolcapone/Entacapone to Cause IDILI.

We have developed an animal model of amodiaquine-induced liver injury that has characteristics very similar to idiosyncratic drug-induced liver injury (IDILI) in humans by impairing immune tolerance using a PD1-/- mouse and cotreatment with anti-CTLA-4. In order to test the usefulness of this model as a general model for human IDILI risk, pairs of drugs with similar structures were tested, one of which is associated with a relatively high risk of IDILI and the other not. One such pair is troglitazone and pioglitazone; troglitazone has caused fatal cases of IDILI while pioglitazone is quite safe. Another pair is tolcapone and entacapone; tolcapone can cause serious IDILI; in contrast, although entacapone has been reported to cause liver injury, it is relatively safe. PD1-/- mice treated with anti-CTLA-4 and troglitazone or tolcapone displayed liver injury as determined by ALT levels and histology, while pioglitazone and entacapone showed less signs of liver injury. One possible mechanism by which drugs could induce an immune response leading to IDILI is by causing the release of danger-associated molecular pattern molecules that activate inflammasomes. We found that the supernatants from incubations of troglitazone, tolcapone, or entacapone with hepatocytes were also able to activate inflammasomes in macrophages, while the supernatant from pioglitazone incubations did not. These results are consistent with an immune mechanism for troglitazone- and tolcapone-induced IDILI and add to the evidence that this may be a general model for IDILI.

Effects of immunization and checkpoint inhibition on amodiaquine-induced liver injury.

If idiosyncratic drug-induced liver injury (IDILI) is immune-mediated, it is possible that an individual's prior exposure to antigens may affect their susceptibility to IDILI. An individual's repertoire of memory immune cells is shaped by every past exposure to antigens. Subsequent drug-induced adverse drug reactions may therefore involve an immune cell's cross reactivity between a prior antigen and resulting drug-modified proteins. Therefore in this experiment, mice were immunized with amodiaquine (AQ)-modified hepatic proteins to mimic a previous exposure; treated with a RIBI adjuvant and anti-CD40 antibodies to stimulate an immune response; and, treated with anti-PD1 and anti-CTLA-4 antibodies prior to AQ treatment in order to overcome immune tolerance. This treatment led to greater liver injury than treatment with AQ alone. However, the mice did not develop serious liver injury. PD1-/- mice were then immunized and treated with AQ and anti-CTLA-4 antibodies so that immune tolerance would be impaired, both during immunization and also during AQ treatment. However, even this did not result in liver failure, and the liver injury was not significantly increased relative to un-immunized PD1-/- mice treated with anti-CTLA-4 and AQ. From these results we conclude that, although previous antigen exposure may affect the risk of IDILI, it appears that a very strong stimulus is required, and impairing immune tolerance remains the most effective method for producing an animal model of IDILI.

Immune mechanisms of idiosyncratic drug-induced liver injury.

Idiosyncratic drug reactions (IDRs) continue to be an important issue. Specifically, idiosyncratic drug-induced liver injury (IDILI) is the most likely IDR to lead to drug withdrawal, and it accounts for a significant portion of all cases of acute liver failure. In addition, IDRs are unpredictable and their mechanisms are not well understood. There is increasing clinical evidence that most IDILI is immune mediated. Several immune mediated mechanistic hypotheses exist such as the hapten and danger hypothesis; however, they do not completely explain the idiosyncratic nature of these reactions. Extensive mechanistic studies are needed to better understand these reactions; however, it is impossible to do controlled experiments in humans, and previous animal models did not properly model IDILI. If IDILI is immune mediated and the major factor preventing liver injury in patients is immune tolerance, then a plausible method to develop an animal model of IDILI would be to impair immune tolerance. This hypothesis has shown promise in developing valid animal models of IDILI as demonstrated by a halothane induced liver injury mouse model developed by depleting myeloid derived suppressor cells (MDSCs), as well as an amodiaquine-, isoniazid-and nevirapine-induced liver injury mouse model developed by impairing immune tolerance by blocking PD-1 and CTLA-4, two immune checkpoint inhibitors. Further characterization and validation of these models is required; however, it is likely that they will make it possible to perform mechanistic studies that have been impossible in the past. Relevance for patients: Idiosyncratic drug-induced liver injury can be serious leading to liver transplantation or death. Their idiosyncratic nature makes mechanistic studies very difficult. However, with the development of the first animal model that is similar to the liver injury that occurs in humans, it will be possible to study the mechanisms involved. With a better mechanistic understanding it should be possible to test drug candidates and produce safer drugs. In addition, it should be possible to design better treatments when drug-induced liver injury does occur.

The Combination of Anti-CTLA-4 and PD1-/- Mice Unmasks the Potential of Isoniazid and Nevirapine To Cause Liver Injury.

Our laboratory recently reported what we believe is the first valid animal model of idiosyncratic drug-induced liver injury (IDILI) by treating PD1-/- mice with an anti-CTLA-4 antibody and amodiaquine (AQ). PD1 and CTLA-4 are important immune checkpoint receptors that are involved in inducing immune tolerance. This model was able to produce significant liver injury that looks very similar to the liver injury seen in humans. Although this model was shown to work with AQ, the question becomes whether blocking immune tolerance would unmask the potential of other drugs to cause IDILI. In this study, we tested isoniazid and nevirapine, both drugs with significant histories of causing IDILI in humans even though they do not cause significant injury in animals with doses that result in therapeutic blood levels. Both drugs in combination with these immune checkpoint inhibitors caused mild but significant delayed onset liver injury, which is similar to the mild injury that they can cause in humans. INH-induced liver injury in this model was associated with an increase in NK cells, while NVP-induced liver injury was associated with a greater increase in CD8 T cells. Although the liver injury caused by these drugs in this model was mild, these results suggest that impairing immune tolerance may be a general method for unmasking the potential of drugs to cause IDILI and therefore provide a screening tool for drug development.

The Role of CD8 T Cells in Amodiaquine-Induced Liver Injury in PD1-/- Mice Cotreated with Anti-CTLA-4.

The mechanism of idiosyncratic drug-induced liver injury (IDILI) remains poorly understood, in part due to the lack of a valid animal model. Clinical evidence suggests that most IDILI is immune mediated, and the major factor preventing liver injury in most patients is immune tolerance. Many attempts have been made in the past to develop an animal model of IDILI, but none had characteristics similar to those of IDILI in humans, and presumably they involved a different mechanism. Recently our laboratory reported a model of amodiaquine (AQ)-induced IDILI using PD1-/- mice and an anti-CTLA4 antibody. This may be the first valid animal model of IDILI because it mimics the characteristics of IDILI in humans. The current study extended the duration of AQ treatment to see if this model would lead to liver failure and to further characterize the associated immune response. Although AQ treatment was extended to 10 weeks and total bilirubin levels were significantly elevated compared to control, there was no further increase from weeks 7 to 10, and the animals did not develop overt liver failure. Mice treated with AQ and anti-CTLA4 had a significant increase in percentage of hepatic CD4, CD8, Th17, and Treg cells after 10 weeks of AQ treatment, as well as significantly decreased NK cells. CD8 T cells have been implicated in several serious idiosyncratic drug reactions, and we used an anti-CD8 antibody to deplete CD8 T cells to study their involvement in this liver injury. We found that depletion of CD8 T cells protected mice from AQ-induced liver injury in this model, which strongly suggests that they are responsible for the liver damage. This is consistent with the finding of CD8 T cells in liver biopsies of human IDILI and may lead the way to an effective treatment for serious IDILI.

Immunization with amodiaquine-modified hepatic proteins prevents amodiaquine-induced liver injury.

Amodiaquine (AQ) has been reported to cause severe idiosyncratic drug-induced liver injury (IDILI) in humans. There is evidence that AQ-induced idiosyncratic drug reactions are immune mediated, but their exact mechanism is not fully understood. AQ is oxidized to a reactive quinoneimine metabolite, and it has been suggested that covalent binding of this metabolite leads to an immune response and liver injury. A valid animal model would greatly facilitate mechanistic studies. This laboratory had previously reported that chronic treatment of C57BL/6 mice with AQ induced a delayed-onset mild liver injury that appeared to resolve with immune tolerance. The current study attempted to prevent immune tolerance by first immunizing mice with AQ-modified hepatic proteins. This study used a soluble adjuvant known as 'covax' that has been reported to produce a better immune response than Freund's adjuvant. After immunization, the mice were treated with 0.2% AQ in food for 5 weeks, as previously done. Paradoxically, the immunization protected animals from AQ-induced liver injury instead of exacerbating it. Consistent with this protection, immunization also appeared to lead to a tolerogenic response with an increase in myeloid derived suppressor cells, M2 macrophages, and FoxP3(+) T-cells. This attempt to develop an animal model of IDILI was unsuccessful and illustrated the complexity of the immune response and the difficulty of inducing a sustained immune response in the liver. It also reinforces the hypothesis that the key determinant of IDILI is immune tolerance.