Supernatant from Hepatocyte Cultures with Drugs That Cause

May 19, 2017 - There is increasing evidence that most idiosyncratic drug-induced liver injury (IDILI) is immune mediated, and in most cases, reactive ...
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Supernatant from Hepatocyte Cultures with Drugs That Cause Idiosyncratic Liver Injury Activates Macrophage Inflammasomes Ryuji Kato†,‡ and Jack Uetrecht*,† †

Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, Toronto, Ontario M5S 3M2, Canada Laboratory of Cardiovascular Pharmacotherapy and Toxicology, Osaka University of Pharmaceutical Sciences, Osaka 569-1094, Japan

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ABSTRACT: There is increasing evidence that most idiosyncratic drug-induced liver injury (IDILI) is immune mediated, and in most cases, reactive metabolites appear to be responsible for the induction of this immune response. Reactive metabolites can cause cell damage with the release of damage-associated molecular patterns (DAMPs), which is thought to be involved in immune activation. Presumably, the reason that the liver is a common target of idiosyncratic drug reactions is because it is the major site of drug metabolism and reactive metabolite formation. Inflammasomes can be activated by DAMPs, and this may be a common mechanism by which DAMPs initiate an immune response. In this study, we tested the ability of drugs to induce the release of DAMPs that activate inflammasomes. The drugs tested were amodiaquine and nevirapine; both are associated with significant incidences of severe IDILI. The hepatocytes were a human hepatocarcinoma functional liver cell-4 (FLC-4) cell line. For the detection of inflammasome activation, we used the human macrophage cell line, THP-1 cells. We found that the supernatant from the incubation of both drugs with FLC-4 cells for 7 days led to increased caspase-1 activity and production of IL-1β by THP-1 cells. However, amodiaquine alone also directly activated THP-1 cells. This is presumably because the myeloperoxidase in THP-1 cells can bioactivate amodiaquine to a reactive metabolite. In contrast, nevirapine requires cytochromes P450 for reactive metabolite formation and therefore required incubation with hepatocytes. These results support the hypothesis that reactive metabolites of drugs can cause the release of DAMPs, which in turn can activate inflammasomes. Inflammasome activation may be an important step in the activation of the immune system by drugs, which in some patients can lead to IDILI. Our in vitro model is simple and convenient for evaluating inflammasome activation, and this may be a method to screen drugs for IDILI risk.

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INTRODUCTION Idiosyncratic drug-induced liver injury (IDILI) remains a serious problem, and it significantly adds to the cost of drug development because it is presently impossible to predict which drug candidates may have to be withdrawn because of IDILI.1 There is increasing evidence that most IDILI is immune mediated, and in most cases, the immune response is initiated by a reactive metabolite.2 Several mechanisms have been proposed for how reactive metabolites can induce an immune response leading to an idiosyncratic drug reaction, but the dominant complementary hypotheses are the hapten and danger hypotheses.2 In general, foreign proteins do not induce a strong immune response; thus, a second signal mediated by costimulatory molecules on antigen presenting cells (APCs) is required.3 Reactive metabolites can cause cell damage leading to the release of damage-associated molecular patterns (DAMPs). It has been shown that treatment of hepatocytes with acetaminophen, which causes liver injury although not IDILI, leads to the release of DAMPs such as high mobility group box-1 (HMGB-1), and the supernatant from these cells led to the activation of macrophages.4 DAMPs act through receptors such © 2017 American Chemical Society

as toll like receptors (TLR) and the receptor for advanced glycation end products (RAGE). These receptors can lead to the activation of inflammasomes, and this may be a common mechanism by which DAMPs activate APCs.5 For example, it is known that mice deficient in NACHT-LRR- and pyrin (NALP), a component of the inflammasome, are resistant to contact hypersensitivity caused by reactive chemicals.6 In addition, we have shown that chemically reactive drugs that cause serious skin rashes activate inflammasomes, while similar reactive drugs that are not associated with serious skin rashes do not.7 By analogy, activation of inflammasomes may be an important mechanism by which chemically reactive species induce an immune response that in some patients leads to IDILI. However, APCs and other macrophages have little P450, and most reactive metabolite formation occurs in hepatocytes. On the other hand, most inflammasome activity is present in macrophages. Therefore, to test whether reactive metabolites formed by hepatocytes lead to inflammasome Received: March 7, 2017 Published: May 19, 2017 1327

DOI: 10.1021/acs.chemrestox.7b00065 Chem. Res. Toxicol. 2017, 30, 1327−1332

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Chemical Research in Toxicology

THP-1 cells within these concentrations (FLC-4 cells, 97.1−114.3%; THP-1 cells, 100.8−117.0%). ATP (5 mM) was used as a positive control for inflammasome activation.2,5 1-Phenyl-1-hexanol (100 μM) was used to inhibit sulfotransferase activity, and 1-aminobenzotriazole (1 mM) was used to inhibit cytochromes P450 activities.20,21 Benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (ZVAD, 10 μg/mL; InvivoGen; CA, USA) was used to inhibit caspase activity. Western Blotting. Protein samples from lysed cells (30 μg) were loaded onto an 8% sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) gel. The resolved proteins were then electrotransferred onto a nitrocellulose membrane (0.2 μm, BioRad, Mississauga, ON, Canada). Rabbit antiamodiaquine primary antibody and rabbit antinevirapine primary antibody were developed as previously described.22,23 They were used as the primary antibody and were detected by goat antirabbit IgG-peroxidase (Sigma-Aldrich Co.). Bound peroxidase was visualized by using Supersignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). Each Western blot was repeated three times. Measurement of IL-1β Concentration in Culture Medium. The culture medium from differentiated THP-1 cells was collected and stored at −80 °C until analysis. IL-1β was measured in each culture medium sample using an ELISA kit (Thermo Fisher Scientific). Caspase-1 Activity of Differentiated THP-1 Cells. Differentiated THP-1 cells (4 × 105 cells/mL) were cultured with FLC-4 culture medium for 18 h in a 24-well plate. Caspase-1 activity was measured using the Caspase-Glo 1 Inflammasome Assay (Promega Corporation, Madison, WI). Caspase-Glo reagent was added to each well and then incubated for 1 h at room temperature. Supernatant from the THP-1 cells (200 μL) was transferred to a 96-well white plate, and the luminescence was measured with a plate reader. Data Analysis. Results are expressed as means ± SD. Statistical analyses were performed using the Tukey multiple comparison tests. P < 0.05 was considered statistically significant.

activation it is necessary to use a combination of hepatocytes and macrophages. We used two drugs, amodiaquine and nevirapine, to test the hypothesis that drugs that cause IDILI initiate an immune response through inflammasome activation. These two drugs are associated with a relatively high incidence of IDILI. Amodiaquine is no longer used for malaria prophylaxis because it is associated with both severe liver injury and agranulocytosis.8 Nevirapine is associated with a significant incidence of serious skin rashes and/or liver toxicity.9 In previous studies, we found that both drugs covalently bind to hepatic proteins in mice.10−12 Amodiaquine is readily oxidized to a reactive iminoquinone by both cytochromes P450 and myeloperoxidase. In contrast, nevirapine requires cytochromes P450 to be oxidized to a reactive quinone methide. Immortalized hepatocyte cell lines and primary isolated liver cells are currently the most widely used for bioactivation experiments;13 however, immortalized cell lines often lack liverspecific functions including the major P450s.14 Human primary hepatocytes are the standard against which other cells are compared for xenobiotic metabolism and toxicity studies.15,16 However, they are in limited supply and also rapidly lose their metabolic capacity. In previous studies, we utilized three-dimensional (3D) cultures of a human functional liver cell (FLC)-4 cell line, which synthesize and secrete albumin, alpha-fetoprotein, and other liver-specific proteins.17 These cells also have significant drug-metabolizing capacity, and they were used in this study to produce reactive metabolites of amodiaquine and nevirapine. We used THP-1 cells, which are a human cell line for the detection of inflammasome activation. These cells have the potential to bioactivate amodiaquine, but it is unlikely that they could bioactivate nevirapine.

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RESULTS IL-1β Production and Caspase-1 Levels in THP-1 Cells Incubated with Amodiaquine or the Supernatants from FLC-4 Cells Incubated with Amodiaquine. In this study, IL-1β production by differentiated THP-1 cells was increased by the supernatant from FLC-4 cells incubated with 3 μM amodiaquine (Figure 1B). However, incubation of THP-1 cells with 3 μM amodiaquine alone also led to an increase in IL-1β (Figure 1A). IL-1β Production and Caspase-1 Levels in THP-1 Cells Incubated with Nevirapine or the Supernatants from FLC-4 Cells Incubated with Nevirapine. Incubation of THP-1 cells with the supernatant from an incubation of FLC-4 cells with nevirapine also led to an increase in IL-1β production, and the production of IL-1β was inhibited by adding the P450 inhibitor 1-aminobenzotriazole to the hepatocyte culture but not by the sulfotransferase inhibitor 1-phenyl-1-hexanol (Figure 2B). However, in this case incubation with nevirapine alone did not lead to an increase in the production of IL-1β (Figure 2A). Covalent Binding of Amodiaquine or Nevirapine to THP-1 or FLC-4 Cell Proteins. As seen in Figure 3, there was covalent binding of amodiaquine to FLC-4 cells (B) but also to THP-1 cells (A), while there was no significant nonspecific binding of the antiamodiaquine antibody in the control blots from incubations without drug. In contrast to amodiaquine, there was no significant covalent binding of nevirapine to THP-1 cell proteins (Figure 4A). There was covalent binding of nevirapine to FLC-4 cell proteins, and the binding was inhibited by adding 1-aminobenzotriazole (Figure 4B). Caspase-1 Levels in THP-1 Cells Incubated with Amodiaquine or the Supernatants from FLC-4 Cells Incubated with Amodiaquine or Nevirapine. The increase in IL-1β release with inflammasome activation is mediated by

MATERIALS AND METHODS

Reagents. Amodiaquine hydrochloride was purchased from Ipca Laboratories Ltd. (Mumbai, India). Nevirapine was kindly supplied by Boehringer-Ingelheim Pharmaceuticals, Inc. (Ridgefield, CT). 1-Phenyl-1-hexanol was obtained from Tokyo Chemical Industry (Tokyo, Japan), and 1-aminobenzotriazole was obtained from SigmaAldrich Co. (Oakville, ON). Other reagents and solvents were commercially available extra-pure grade chemicals. Cell Cultures. FLC-4 cells (previous names, JHH-4 (JCRB0435)18) were obtained from Health Science Research Resources Bank (Osaka, Japan). THP-1 cells (JCRB0112) were obtained from Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). FLC-4 cells (passage 20−60) were grown and maintained in Dulbecco’s modified Eagle’s medium (DMEM, 4.5 g glucose/L, Thermo Fisher Scientific, Waltham, MA), and THP-1 cells (passage 20−60) were grown and maintained in Roswell Park Memorial Institute medium (RPMI, Thermo Fisher Scientific), containing 10% fetal bovine serum (Sigma-Aldrich Co.) at 37 °C with 5% CO2 in 25 cm2 tissue culture flasks (Becton-Dickinson Ind., Mississauga, ON). A FLC-4 cell suspension (100 μL, 1.5 × 104 cells/mL) and the same volume of medium were applied to a Prime Surface 96U plate (S-BIO, Hudson, NH) for 3D culture. The cells in the 96U plate were cultured at 37 °C with 5% CO2 in the presence of amodiaquine or nevirapine for 7 days. THP-1 cells (4 × 105 cells/mL) were differentiated in medium containing phorbol 12-myristate 13-acetate (50 ng/mL, Sigma-Aldrich Co.) for 3 days in a 24-well multiplate. On the fourth day, each well was washed with PBS (Sigma-Aldrich Co.), medium (1 mL) was added to each well, and the cells were incubated at 37 °C with 5% CO2 for 24 h. Then, the medium was aspirated, and culture medium from the FLC-4 cells that had been cultured with drugs for 7 days was added and incubated at 37 °C with 5% CO2 for 18 h. The drug concentrations used in this study were similar to their therapeutic concentrations (amodiaquine, 0.3−3 μM; nevirapine, 10−100 μM).9,19 There was no effect on the viability of FLC-4 and 1328

DOI: 10.1021/acs.chemrestox.7b00065 Chem. Res. Toxicol. 2017, 30, 1327−1332

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Figure 1. Incubation of THP-1 cells with amodiaquine or the supernatant from the incubation of hepatocytes with amodiaquine led to the release of IL-1β. (A) Levels of IL-1β secreted by THP-1-derived macrophages in response to 18 h of treatment with amodiaquine itself or (B) the supernatant from an incubation of hepatocytes with amodiaquine for 7 days, with or without a caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (ZVAD). Statistical significance was determined using the one-way ANOVA, where **, p < 0.01, n = 3−5.

Figure 2. Incubation of THP-1 cells with nevirapine did not lead to IL-1β release, but incubation with the supernatant from hepatocytes incubated with nevirapine did. (A) Levels of IL-1β secreted by THP-1-derived macrophages incubated for 18 h with nevirapine itself or (B) with the supernatant from an incubation of hepatocytes with nevirapine for 7 days, with and without a sulfotransferase inhibitor (1-phenyl-1-hexanol), a cytochromes P450 inhibitor (1-aminobenzotriazole), or a caspase inhibitor (benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, ZVAD). Statistical significance was determined using one-way ANOVA, where **p < 0.01, n = 3−5.

activated caspase-1, and we added the caspase inhibitor, ZVAD, to control incubations to ensure that this was the mechanism for the increase in IL-1β. Caspase-1 activities in differentiated THP-1 cells was increased by the supernatant from FLC-4 cells incubated with 3 μM amodiaquine (Figure 5B). However, incubation of THP-1 cells with 1 and 3 μM amodiaquine alone also led to an increase in

caspase-1 activity (Figure 5A). Incubation of THP-1 cells with the supernatant from an incubation of FLC-4 cells with nevirapine led to an increase in caspase-1 activity (Figure 5C). In preliminary experiments, we tested whether there was an increase in the level of HMGB-1 in the supernatant from FLC-4 cells incubated with nevirapine analogous to the results 1329

DOI: 10.1021/acs.chemrestox.7b00065 Chem. Res. Toxicol. 2017, 30, 1327−1332

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Figure 3. Covalent binding of amodiaquine to proteins from THP-1 cells or hepatocytes. (A) Western blot analysis of proteins from THP-1-derived macrophages incubated for 18 h with amodiaquine (3 μM) and (B) proteins from FLC-4 cells incubated for 7 days with amodiaquine (3 μM). Protein loading was 30 μg per lane for all blots; the primary antiamodiaquine antiserum was diluted 1:2000.

Figure 4. Nevirapine covalently binds to hepatocyte proteins but not THP-1 cell proteins. (A) Western blot analysis of proteins from THP-1-derived macrophages incubated for 18 h with nevirapine (100 μM) and (B) proteins from FLC-4 cells incubated for 7 days with nevirapine (100 μM) with and without a cytochromes P450 inhibitor, 1-aminobenzotriazole. Protein loading was 30 μg per lane for all blots; the primary antinevirapine antiserum was diluted 1:500.

reactive drugs that cause idiosyncratic reactions from drugs with similar structures that do not. However, in the case of IDILI, reactive metabolites of drugs are formed in hepatocytes, but most of the inflammasome activity is in macrophages. However, if the reactive metabolites cause cell injury with the release of DAMPs, the DAMPs may, in turn, lead to inflammasome activation. The activation of inflammasomes activates caspase-1, and active caspase-1 then converts the pro-IL-1β and pro-IL-18 into their bioactive, mature forms. IL-1β is a strong pro-inflammatory molecule and promotes an immune response.24,25 Therefore, inflammasome activation may be an important bridge between reactive metabolite formation and the induction of an immune response. In this study, we tested the hypothesis that drugs that cause IDILI cause the release of DAMPs from hepatocytes leading to inflammasome activation in macrophages.

of Martin-Murphy et al., but no significant increase was detected (data not shown). There are several different forms of HMGB-1 based on oxidation state and degree of acetylation and methylation; therefore, it is possible that although the total amount of HMGB-1 was not increased by incubation with nevirapine there was an increase in one of the more inflammatory forms of the protein. There are many other possible DAMPs, and future studies will attempt to identify the putative DAMPs produced by the FLC-4 cells.

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DISCUSSION

If most IDILI is immune mediated and caused by reactive metabolites, it raises the question of how reactive metabolites induce an immune response. As mentioned in the Introduction, it appears as if inflammasome activation may differentiate chemically 1330

DOI: 10.1021/acs.chemrestox.7b00065 Chem. Res. Toxicol. 2017, 30, 1327−1332

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Chemical Research in Toxicology

Figure 5. An increase in caspase-1 activity correlates with the release of IL-1β. (A) Caspase-1 activity of THP-1-derived macrophages in response to incubation for 18 h with amodiaquine itself or (B) the supernatant from incubation of hepatocytes with amodiaquine, or (C) supernatant from the incubation of hepatocytes with nevirapine, with or without a caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (ZVAD). Statistical significance was determined using one-way ANOVA, where *, p < 0.05, and **, p < 0.01, n = 3.

DAMPs, and that these DAMPs activate inflammasomes leading to an immune response. The combination of a 3D culture of FLC-4 cells and differentiated THP-1 cells is a convenient system to test drugs for their ability to activate inflammasomes, and this may be useful for screening drug candidates for IDILI potential. Obviously, more drugs will need to be tested to determine how accurate this assay may be.

Other studies have examined the ability of supernatants from hepatocytes that have been incubated with drugs to activate macrophages.26,27 In the study by Oda et al., many drugs were tested, and the amount of various cytokine mRNAs in the HL-60 cells (a human promyelocytic cell line) were quantified.27 In that study, using a nevirapine concentration above the therapeutic concentration, they did not detect a significant increase in IL-1β mRNA. In contrast, we found that the supernatant from the incubation of nevirapine with FLC-4 cells at a therapeutic concentration led to the activation of inflammasomes in THP-1 cells. Nevirapine can be bioactivated to a reactive quinone methide and a benzylic sulfate.28 The quinone methide is very reactive and binds to P450s leading to their inactivation;23 therefore, it is extremely unlikely that the supernatant from hepatocytes would contain any quinone methide. In contrast, the benzylic sulfate is much less reactive and could to be present in the FLC-4 cell supernatant. However, we did not detect significant amounts of the sulfate in the supernatant by LC/MS (data not shown). Production of IL-1β was inhibited by adding the cytochromes P450 inhibitor (1-aminobenzotriazole) to the FLC-4 culture but not by a sulfotransferase inhibitor (1-phenyl-1-hexanol). Therefore, we conclude that the quinone methide caused the release of DAMPs from the hepatocytes, and it was these DAMPs that were responsible for inflammasome activation in THP-1 cells. We also found that the supernatant from the incubation of amodiaquine with FLC-4 cells at therapeutic concentrations led to the activation of inflammasomes in THP-1 cells. However, amodiaquine is bioactivated by THP-1 cells leading to covalent binding to the cells, and it directly activated THP-1 cells; therefore, we cannot tell whether it was DAMPs or amodiaquine in the FLC-4 cell supernatant that led to inflammasome activation in THP-1 cells. Unlike nevirapine, which requires cytochromes P450 for reactive metabolite formation, amodiaquine is also oxidized to a reactive iminoquinone by myeloperoxidase, which covalently binds to bone marrow cells, and this is presumably why it can also cause agranulocytosis.22 In conclusion, our results are consistent with the hypothesis that the mechanism of IDILI, at least for some drugs, involves oxidation to a reactive metabolite by hepatocytes, which release

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AUTHOR INFORMATION

Corresponding Author

*Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, Ontario M5S 3M2, Canada. Tel: 416-978-8939. Fax: 416-978-8939. E-mail: jack.uetrecht@ utoronto.ca. ORCID

Jack Uetrecht: 0000-0003-1024-1302 Funding

This work was supported by grants from the Canadian Institutes of Health Research. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS R.K. is a trainee from the Japanese Society of Clinical Pharmacology and Therapeutics. We thank Kyle Weston and Yanshan Cao for technical assistance in performing the experiments.

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ABBREVIATIONS APCs, antigen presenting cells; DAMPs, damage leading to the release of damage-associated molecular patterns; DMEM, Dulbecco’s modified Eagle’s medium; FLC, functional liver cell; HMGB-1, high mobility group box-1; IDILI, idiosyncratic drug-induced liver injury; IL, interleukin; NALP, NACHT, LRR, and PYD domain-containing protein; RAGE, receptor for advanced glycation end products; RPMI, Roswell Park Memorial Institute medium; 3D, three-dimensional; TLR, toll like receptors; ZVAD, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone 1331

DOI: 10.1021/acs.chemrestox.7b00065 Chem. Res. Toxicol. 2017, 30, 1327−1332

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(22) Lobach, A. R., and Uetrecht, J. (2014) Involvement of myeloperoxidase and NADPH oxidase in the covalent binding of amodiaquine and clozapine to neutrophils: implications for druginduced agranulocytosis. Chem. Res. Toxicol. 27, 699−709. (23) Sharma, A. M., Klarskov, K., and Uetrecht, J. (2013) Nevirapine bioactivation and covalent binding in the skin. Chem. Res. Toxicol. 26, 410−421. (24) Heymann, F., and Tacke, F. (2016) Immunology in the liver– from homeostasis to disease. Nat. Rev. Gastroenterol. Hepatol. 13, 88− 110. (25) Guo, H., Callaway, J. B., and Ting, J. P. (2015) Inflammasomes: mechanism of action, role in disease, and therapeutics. Nat. Med. 21, 677−687. (26) Kegel, V., Pfeiffer, E., Burkhardt, B., Liu, J. L., Zeilinger, K., Nüssler, A. K., Seehofer, D., and Damm, G. (2015) Subtoxic Concentrations of Hepatotoxic Drugs Lead to Kupffer Cell Activation in a Human In Vitro Liver Model: An Approach to Study DILI. Mediators Inflammation 2015, 640631. (27) Oda, S., Matsuo, K., Nakajima, A., and Yokoi, T. (2016) A novel cell-based assay for the evaluation of immune- and inflammatory-related gene expression as biomarkers for the risk assessment of drug-induced liver injury. Toxicol. Lett. 241, 60−70. (28) Pinheiro, P. F., Pereira, S. A., Harjivan, S. G., Martins, I. L., Marinho, A. T., Cipriano, M., Jacob, C. C., Oliveira, N. G., Castro, M. F., Marques, M. M., Antunes, A. M., and Miranda, J. P. (2017) Hepatocyte spheroids as a competent in vitro system for drug biotransformation studies: nevirapine as a bioactivation case study. Arch. Toxicol. 91, 1199− 1211.

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DOI: 10.1021/acs.chemrestox.7b00065 Chem. Res. Toxicol. 2017, 30, 1327−1332