Diagnostic significance and utility of circulating redox biomarkers in patients with gastric cancer

doi:10.21203/rs.3.rs-2755112/v1

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Diagnostic significance and utility of circulating redox biomarkers in patients with gastric cancer

https://doi.org/10.21203/rs.3.rs-2755112/v1

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The aim of our study was to evaluate the redox status, antioxidant barrier as well oxidative damages in gastric cancer patients. We are also the first to assess the diagnostic utility of redox parameters in GC patients in respect to histopathological parameters. 50 patients with gastric cancer and 50 healthy controls matched for sex and age were included in the study. Antioxidant barrier, redox status and oxidative damage products were measured in serum/plasma samples using colorimetric or spectrophotometric method. The activity of SOD (p < 0.05) was significantly higher whereas the activity of CAT (p < 0.0001), GPx (p < 0.0001), GR (p < 0.0001) and GSH (p < 0.05) was considerably lower in GC patients compared to the control group. Levels of TOS (p < 0.0001), OSI (p < 0.0001), AOPP (p < 0.0001), IMA (p < 0.01), LOOH (p < 0.0001), 8-Iso-P (p < 0.0001) and DNA/RNA (p < 0.0001) were significantly higher, and the levels of TAC (p < 0.0001) and total thiols (p < 0.0001) were considerably lower in GC patients compared to the healthy control. Some redox parameters are characterised by high AUC value in differentiation GC patients according to histopathological parameters. Gastric cancer is strongly linked to systemic redox imbalance as well as increased oxidative damage to proteins, lipids and DNA. Redox biomarkers can be potential diagnostic indicators of gastric cancer advancement.

Health sciences/Biomarkers/Diagnostic markers

Health sciences/Oncology/Cancer/Gastrointestinal cancer

Health sciences/Oncology/Cancer/Gastrointestinal cancer/Gastric cancer

According to the Global Cancer Observatory (GLOBOCAN) data from 2020, gastric cancer (GC) was classified as 4th most common type of cancer in the world among men and 7th most frequent one in women. Moreover, GC is responsible for 7.7% of all cancer deaths which places it 4th in terms of mortality among all cancers. Despite the fact that in recent decades the incidence and mortality due to gastric cancer decreased, GLOBOCAN prognosis that in 2040 the number of new cases of gastric cancer will increase from 1.09 in 2020 to over 1.8 million in 2040 and the number of deaths will come from 769,000 to 1.27 million (Ferlay et al). Moreover, about 95% of all gastric cancer cases are diagnosed in patients over 40 years old but in recent years a considerable increase in the incidence in the group of young people under 40 years of age was observed ^2,3. Therefore, scientists focus on risk factors and mechanisms participating in gastric cancer development especially in the young. It is well known that gastric carcinogenesis is closely linked to Helicobacter pylori infection, inherited cancer predisposition syndromes eg. Lynch syndrome, juvenile polyposis syndrome (JPS) or Peutz-Jeghers syndrome; lifestyle, psychosocial environment, socioeconomic status, and dietary habits ². Lifestyle and dietary habits like stress, smoking, drinking alcohol and high-fat or high-carbohydrate diets may also be responsible for the overproduction of reactive oxygen species (ROS) which leads to the development of oxidative stress in the case of an ineffective antioxidant barrier ⁴ Although ROS in physiological concentrations act as regulators of cellular signal transduction and energy transmission, imbalance between the generating and scavenging capability of ROS may result in cellular damages. ROS can react with proteins and lipids transforming them into oxidised forms, or by damaging the structure of DNA and tumour suppressor genes promoting the carcinogenesis ^5,6. It has been observed that oxidative stress-mediated signalling affects all features of cancer cells behaviour like cell cycle progression and proliferation, cell morphology and survival, apoptosis, cell-cell adhesion, cell motility or energy metabolism. ROS through activation of transcription factors, including NF-κB, activator protein 1 (AP1), and hypoxia inducible factor 1α (HIF1α) take part in regulating expression of genes associated with cancer cell growth and survival, angiogenesis, invasion, and metastasis ⁷.

In our previous papers focusing on assessment of oxidative stress in colorectal cancer we demonstrated that CRC is associated with both enzymatic and non-enzymatic redox imbalance as well as increased oxidative damage to proteins and lipids. Moreover, we demonstrated that many redox biomarkers differentiate patients with CRC from healthy individuals with high sensitivity and specificity ^8,9. We also showed that serum catalase (CAT) may be potential non-invasive biomarkers indicating tumour invasion depth ⁸. However, still little is known about the clinical significance and diagnostic utility of oxidative stress/redox parameters in patients with gastric cancer. Therefore, the aim of our study was to evaluate the redox status, enzymatic and non-enzymatic antioxidants as well oxidative damage to proteins, lipids and DNA in gastric cancer patients compared to the healthy controls as well as to assess the diagnostic utility of oxidative stress parameters using the analysis of receiver operating characteristic (ROC) curve. We are also the first to assess the redox parameters in gastric cancer patients in respect to histopathological classifications (TNM, Lauren’s and Goseki’s classification) and histopathological parameters such as histological type, histological differentiation grade as well as presence of vascular or neural infiltration and desmoplasia.

Clinical characteristics

The study included 50 patients with gastric cancer. The moderately differentiated grade (G2) of the tumour was diagnosed in 40% of patients whereas poorly differentiated grade was present in 60% of them. Most of the subjects (68%) had adenocarcinoma, and 32% of patients had mucinous adenocarcinoma. 10.0% of patients had a pT1 stage of tumour, 30% had a pT2, 34% had a pT3 and 26% of patients had a pT4 stage. The percentage of patients without lymph node (N0) and distant metastasis (M0) was 24% and 66%, respectively. Small desmoplasia was present in 70% of patients and diffuse in 30%. Detailed characteristics of the study groups are summarised in Table 1.

Table 1

Characteristics of study group.
Parameter	n (%)
Age <60 >60	10 (20.0%) 40 (80.0%)
Sex male female	33 (66.0%) 17 (34.0%)
Tumour’s diameter ≤5cm >5cm	14 (28.0%) 36 (72.0%)
Tumor location Upper 1/3 Middle 1/3 Lower 1/3 Whole stomach	17 (34.0%) 15 (30.0%) 10 (20.0%) 8 (16.0%)
Histological type adenocarcinoma adenocarcinoma mucinosum	34 (68.0%) 16 (32.0%)
Histological differentiation grade moderately differentiated poorly differentiated	20 (40.0%) 30 (60.0%)
Depth of invasion T1 T2 T3 T4	5 (10.0%) 15 (30.0%) 17 (34.0%) 13 (26.0%)
Lymph node metastasis N0 N1 + N2	12 (24.0%) 38 (76.0%)
Distant metastasis M0 M1 + M2	33 (66.0%) 17 (34.0%)
Vascular infiltration absent present	12 (24.0%) 38 (76.0%)
Neural infiltration absent present	14 (28.0%) 36 (72.0%)
Desmoplasia small diffuse	35 (70.0%) 15 (30.0%)
Lauren’s classification intestinal diffuse	21 (42.0%) 29 (48.0%)
Goseki classification I II III IV	6 (12.0%) 19 (38.0%) 11 (22.0%) 14 (28.0%)

Antioxidant Defence

In order to evaluate the antioxidant barrier, we measured the activity of antioxidant enzymes (i.e., Cu,Zn-superoxide dismutase (SOD-1), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR)) and concentration of non-enzymatic antioxidants - glutathione (GSH). The activity of CAT, GPx, GR and GSH was considerably lower whereas SOD activity was significantly higher in the serum of patients with gastric cancer compared to the control group (p < 0.0001, p < 0.0001, p < 0.0001, p < 0.05, p < 0.05 respectively) (Fig. 1). CAT activity was significantly higher and SOD was lower in patients in T1 + T2 stages in comparison with T3 + T4 (p < 0.05, p < 0.05, respectively) (Fig. 4). SOD showed considerably higher activity in patients with distant metastasis compared to those without metastasis (p < 0.05) and CAT in patients with intestinal type of gastric cancer according to Lauren’s classification compared to diffuse type (p < 0.05) and in I + II types of gastric cancer according to Goseki classification than in III + IV (p < 0.01) (Fig. 6, Fig. 7). GPx activity was statistically siginificant increased in patients with adenocarcinoma mucinosum than in adenocarcinoma (p < 0.05) as well as in patients in T1 + T2 stages in comparison with T3 + T4 (p < 0.05) (Fig. 3, Fig. 4). We observed increased GR and GSH activity in patients without lymph node metastasis compared to those with metastasis (p < 0.05, p < 0.05, respectively) (Fig. 5). GSH was also higher in patients without distant metastasis in comparison with metastasis (p < 0.05) (Fig. 5). GPx, GR and GSH level was considerably higher in patients with intestinal type of gastric cancer according to Lauren’s classification compared to diffuse type (p < 0.05, p < 0.05, p < 0.05, respectively) and in patients without vascular infiltration (p < 0.05, p < 0.01, p < 0.001, respectively) (Fig. 6). We demonstrated a significant increase of GSH activity in patients with I + II types of gastric cancer according to Goseki classification than in III + IV (p < 0.05) and in patients without neural invasion (p < 0.001) (Fig. 7).

Total Antioxidant/oxidant Status

To assess the redox status, we determined total antioxidant capacity (TAC), total oxidant status (TOS), and we calculated oxidative stress index (OSI) by dividing TOS level by TAC level (TOS/TAC ratio). In the plasma of patients with gastric cancer, TAC level was significantly lower compared to the control group (p < 0.0001) (Fig. 1). We observed considerable increase in TOS in the plasma of the study group compared to healthy controls (p < 0.0001) (Fig. 1). The OSI value in gastric cancer patients was significantly higher than in the healthy controls (p < 0.0001) (Fig. 1). TOS was considerably higher in patients with G3 grade compared to G2 (p < 0.05) (Fig. 3). TOS and OSI value was also increased in patients with T3 + T4 stages in comparison with T1 + T2 (p < 0.05, p < 0.01 respectively) and in patients with lymph node metastasis (p < 0.01, p < 0.05, respectively) (Fig. 4, Fig. 5). Interestingly, we demonstrated higher levels of TOS in patients with distant metastasis in comparison with those without metastasis (p < 0.05) (Fig. 6). TAC was statistically significantly increased in patients with intestinal type of gastric cancer according to Lauren’s classification compared to diffuse type (p < 0.05) (Fig. 7).

Oxidative Damage Products

To evaluate the oxidative stress, we used oxidative damage products of proteins (total thiols, ischemia-modified albumin (IMA), advanced oxidation protein products (AOPP)), lipids (lipid hydroperoxides (LOOH), 8-Isoprostane (8-Iso-P)) and DNA (DNA/RNA products). There was a statistically significant increase in IMA and AOPP in patients with gastric cancer compared to the control group (p < 0.01, p < 0.0001 respectively). LOOH, 8-Iso-P and DNA/RNA concentrations were also considerably higher (p < 0.0001, p < 0.0001, p < 0.0001, respectively) whereas total thiols were significantly lower in the plasma of the study group than in the control group (p < 0.0001) (Fig. 2). AOPP concentration was significantly higher in patients with tumours with diameter > 5cm than in tumours ≤ 5cm (p < 0.05) (Fig. 3). Levels of AOPP and LOOH were increased in patients with adenocarcinoma mucinosum compared to those with adenocarcinoma (p < 0.01, p < 0.05, respectively) (Fig. 3). We demonstrated higher concentration of total thiols in patients in G3 stage in comparison with G2 (p < 0.05), in patients with lymph node metastasis (p < 0.05) and in patients with III + IV types of gastric cancer according to Goseki classification in comparison with I + II (p < 0.01) (Fig. 3, Fig. 5, Fig. 7). We showed a significant increase in AOPP concentration increased in patients with T3 + T4 stages in comparison with T1 + T2 (p < 0.05), in patients with lymph node metastasis (p < 0.05), in patients with distant metastasis (p < 0.05) and in patients with diffuse type of gastric cancer according to Lauren’s classification compared to intestinal type (p < 0.05) (Fig. 4, Fig. 5, Fig. 6, Fig. 7).

ROC Analysis

ROC analysis was performed to assess the diagnostic value of oxidative stress biomarkers in the diagnostics of gastric cancer (Tables 2–4). We demonstrated that all determined redox parameters differentiate GC patients from healthy controls, to a large extent. We also proved that oxidative stress biomarkers are useful in the differential diagnosis of gastric cancer patients (Table 2). We showed a high diagnostic value of AOPP and LOOH in differentiating the patients with adenocarcinoma from adenocarcinoma mucinosum (AUC for AOPP = 0.7725, for LOOH = 0.7022) (Table 3). GPx, TOS and total thiols proved to be helpful in differentiation moderately differentiated (G2) from poorly differentiated tumours (G3) (AUC for GPx = 0.7067, for TOS = 0.7464, for total thiols = 0.7279) (Table 3). We also demonstrated a very high diagnostic value of SOD (AUC = 0.7273), CAT (AUC = 0.7417), GPx (AUC = 0.6997), TOS (AUC = 0.7451), OSI (AUC = 0.7527) and AOPP (AUC = 0.7284) in differentiating the groups of patients with gastric cancer at stage pT1 + pT2 of tumour invasion from patients with stage pT3 + pT4 (Table 3). Parameters like GR (AUC = 0.7460), GSH (AUC = 0.7619), TOS (AUC = 0.7964), OSI (AUC = 0.7435), AOPP (AUC = 0.7341) and total thiols (AUC = 0.7460) may be useful for differentiation of patients with lymph node metastasis from those without lymph node metastasis (Table 3). SOD (AUC = 0.7190), GSH (AUC = 0.7106), TOS (AUC = 0.7105) and AOPP (AUC = 0.6889) proved to be helpful in differentiation patients with present and absent distant metastasis (Table 3). We showed a high diagnostic value of CAT (AUC = 0.7531), GR (AUC = 0.7022), GPx (AUC = 0.7135), GSH (AUC = 0.6930), TAC (AUC = 0.7135) and AOPP (AUC = 0.7032) in differentiation diffuse type from intestinal type of gastric cancer according to Lauren classification and CAT (AUC = 0.7827), GSH (AUC = 0.7255) and total thiols (AUC = 0.7561) in differentiation I + II types from III + IV types of gastric cancer according to Goseki classification (Table 4). Particular attention should be paid to GR, GPx and GSH for which AUC in the presence of vascular infiltration were 0.8933, 0.8000 and 0.9853 and GSH with AUC = 0.9875 in the presence of neural infiltration (Table 4).

Table 2

Receiver operating characteristic (ROC) analysis of oxidative stress biomarkers of gastric cancer patients and the controls. Abbreviations: SOD – superoxide dismutase-1, CAT – catalase, GPx – glutathione peroxidase, GR – glutathione reductase, GSH – reduced glutathione, TAC – total antioxidant capacity, TOS – total oxidant status, OSI – oxidative stress index, IMA – ischemia-modified albumin, AOPP – advanced oxidation protein products, LOOH – lipid hydroperoxides, 8-Iso-P – 8-Isoprostane.
Parameter	AUC	p-value	Cut-off	Sensitivity (%)	Specificity (%)
Antioxidant defence
SOD (mU/100 mg protein)	0.6896	0.0255	< 241.6	66.67	66.67	0.5304–0.8488
CAT (nmol H₂O₂/100 mg protein)	1.000	< 0.0001	< 115.8	100.0	100.0	1.000–1.000
GPx (mU/100 mg protein)	0.9538	< 0.0001	< 100.2	90.24	92.86	0.8996–1.000
GR (µU/100 mg protein)	0.9977	< 0.0001	< 9.832	95.45	94.87	0.9914–1.000
GSH (µg/100 mg protein)	0.6862	0.0119	< 0.2986	85.37	85.19	0.5498–0.8226
Redox status
TAC (nmol/100 mg protein)	1.000	< 0.0001	< 126.5	100.0	100.0	1.000–1.000
TOS (µmol/100 mg protein)	0.8339	< 0.0001	> 4.257	75.0	74.19	0.7385–0.9293
OSI (TOS/TAC ratio)	0.8954	< 0.0001	> 1.203	78.38	77.42	0.8210–0.9697
Protein, lipid and DNA oxidative damage
Total thiols (µM/mg protein)	1.000	< 0.0001	> 9.528	100.0	100.0	1.000–1.000
IMA (U/mg protein)	0.7277	0.0014	> 0.2761	64.10	62.07	0.5943–0.8610
AOPP (µmol/100 mg protein)	1.000	< 0.0001	> 2.774	100.0	100.0	1.000–1.000
LOOH (nmol/100 mg protein)	0.8864	< 0.0001	> 524.5	87.76	90.00	0.8003–0.9724
8-Iso-P (pg/100 mg protein)	0.9785	< 0.0001	> 89.97	97.78	92.00	0.9369–1.000
DNA/RNA (ng/mg protein)	1.000	< 0.0001	> 1.422	100.0	100.0	1.000–1.000

Table 3

Receiver operating characteristic (ROC) analysis in gastric cancer patients for histological type, histological differentiation grade, depth of tumour invasion, presence of lymph node and distant metastasis. Abbreviations: SOD – superoxide dismutase-1, CAT – catalase, GPx – glutathione peroxidase, GR – glutathione reductase, GSH – reduced glutathione, TOS – total oxidant status, OSI – oxidative stress index, AOPP – advanced oxidation protein products, LOOH – lipid hydroperoxides.
Parameter	AUC	p-value	Cut-off	Sensitivity (%)	Specificity (%)
Histological type
AOPP (µmol/100 mg protein)	0.7725	0.0050	> 15.23	73.33	69.57	0.5986–0.9463
LOOH (nmol/100 mg protein)	0.7022	0.0507	< 1304	63.64	65.52	0.5074–0.8970
		Histological differentiation grade
GPx (mU/100 mg protein)	0.7067	0.0304	> 1.297	64.00	60.00	0.5442–0.8691
TOS (µM/mg protein)	0.7464	0.0181	> 12.46	65.22	66.67	0.5795–0.9133
Total thiols (µM/mg protein)	0.7279	0.0157	> 0.2152	75.00	75.00	0.5627–0.8930
Depth of tumour invasion
SOD (mU/100 mg protein)	0.7273	0.0373	< 2.806	66.67	63.64	0.5369–0.9177
CAT (nmol H₂O₂/100 mg protein)	0.7417	0.0370	< 125.0	77.78	60.00	0.5191–0.9642
GPx (mU/100 mg protein)	0.6997	0.0443	< 1.300	69.23	61.54	0.5165–0.8830
TOS (µM/mg protein)	0.7451	0.0185	> 6.735	75.00	72.73	0.5790–0.9112
OSI (TOS/TAC ratio)	0.7527	0.0079	> 0.211	65.22	62.50	0.5873–0.9181
AOPP (µmol/100 mg protein)	0.7284	0.0243	> 9.532	70.37	66.67	0.5707–0.8861
Presence of lymph node metastasis
GR (µU/100 mg protein)	0.7460	0.0247	< 18.60	70.0	68.00	0.5536–0.9384
GSH (µg/100 mg protein)	0.7619	0.0195	< 10.78	66.67	67.86	0.5792–0.9446
TOS (µM/mg protein)	0.7964	0.0059	> 6.735	75.0	80.00	0.6397–0.9532
OSI (TOS/TAC ratio)	0.7435	0.0192	> 0.509	72.73	75.00	0.5631–0.9239
AOPP (µmol/100 mg protein)	0.7341	0.0367	> 9.238	71.43	77.78	0.5709–0.8973
Total thiols (µM/mg protein)	0.7460	0.0282	> 0.211	67.86	66.67	0.5645–0.9276
		Presence of distant metastasis
SOD (mU/100 mg protein)	0.7190	0.0430	< 2.349	63.64	63.64	0.5438–0.8942
GSH (µg/100 mg protein)	0.7106	0.0269	< 12.73	65.22	68.75	0.5436–0.8776
TOS (µM/mg protein)	0.7105	0.0246	> 15.83	63.16	65.00	0.5441–0.8769
AOPP (µmol/100 mg protein)	0.6889	0.0472	> 12.82	62.50	56.52	0.5136–0.8641

Table 4

Receiver operating characteristic (ROC) analysis in gastric cancer patients for type of GC according to Lauren and Goseki classifications and vascular and neural infiltration. Abbreviations: CAT – catalase, GPx – glutathione peroxidase, GR – glutathione reductase, GSH – reduced glutathione, TAC – total antioxidant capacity, AOPP – advanced oxidation protein products.
Parameter	AUC	p-value	Cut-off	Sensitivity (%)	Specificity (%)
Lauren classification
CAT (nmol H₂O₂/100 mg protein)	0.7531	0.0328	> 97.22	68.75	70.00	0.5663–0.9399
GR (µU/100 mg protein)	0.7022	0.0476	> 18.60	70.59	68.75	0.5124–0.8921
GPx (mU/100 mg protein)	0.7135	0.0265	> 1.305	68.42	72.22	0.5352–0.8917
GSH (µg/100 mg protein)	0.6930	0.0449	> 12.69	63.16	61.11	0.5188–0.8672
TAC (nmol/100 mg protein)	0.7135	0.0265	< 3.524	68.42	66.67	0.5445–0.8824
AOPP (µmol/100 mg protein)	0.7032	0.0347	> 14.00	63.16	66.67	0.5271–0.8793
Goseki classification
CAT (nmol H₂O₂/100 mg protein)	0.7827	0.0043	> 69.44	70.59	61.11	0.6327–0.9327
GSH (µg/100 mg protein)	0.7255	0.0277	> 11.21	72.73	69.23	0.5365–0.9147
Total thiols (µM/mg protein)	0.7561	0.0090	> 21.77	68.18	66.67	0.6009–0.9113
Vascular infiltration
GR (µU/100 mg protein)	0.8933	0.0054	> 14.87	80.0	80.00	0.7824-1.000
GPx (mU/100 mg protein)	0.8000	0.0339	> 1.291	80.0	80.00	0.5390-1.000
GSH (µg/100 mg protein)	0.9853	0.0017	> 7.213	94.12	100.0	0.9491-1.000
		Neural infiltration
GSH (µg/100 mg protein)	0.9875	0.0005	> 7.213	96.88	100.0	0.9569-1.000

Correlations

All statistically significant associations are presented in Fig. 8. We observed positive correlations between sex and AOPP (p = 0.044, R = 0.379) and LOOH level (p = 0.045, R = 0.361).

Patient’s age was negatively correlated with GSH activity (p = 0.018, R=-0.376). Interestingly, we demonstrated a negative correlation between tumour’s location and TAC level (p = 0.047, R=-0.388). LOOH concentration was negatively associated with histological type of the tumour (p = 0.024, R=-0.339). GPx (p = 0.012, R = 0.406), GR (p = 0.033, R = 0.371), GSH (p = 0.038, R = 0.341) and AOPP (p = 0.042, R = 0.299) correlated positively whereas TAC (p = 0.047, R=-0.315) negatively with Lauren’s classification. GPx and total thiols were associated with histological differentiation grade (p = 0.042, R = 0.269; p = 0.044, R = 0.285). GPx was also correlated with depth of tumour invasion (pT) (p = 0.05, R = 0.368). Moreover, the level of GR (p = 0.042, R = 0.299), OSI (p = 0.018, R = 0.375) and AOPP (p = 0.031, R = 0.301) correlated positively with presence of lymph node metastasis (pN). The significant associations were also found between SOD (p = 0.05, R=-0.397), GSH (p = 0.039, R = 0.273), and total thiols concentration (p = 0.036, R = 0.292) and presence of distant metastasis. Positive correlation was also present between GR activity (p = 0.035, R = 0.355) and vascular infiltration. Moreover, there were also statistically significant correlations between vascular and neural infiltration and GSH level (p = 0.037, R = 0.359; p = 0.028, R = 0.484 respectively).

Our study is the first to assess the diagnostic utility of redox biomarkers in patients with gastric cancer. We have shown a decrease in activity of both enzymatic and non-enzymatic antioxidants as well as increased oxidation of proteins, lipids and DNA in the blood of GC patients. Moreover, we showed that some of redox parameters may be helpful and may be novel non-invasive biomarkers in differentiation GC patients according to histological type, histological differentiation grade, tumour invasion depth, the occurrence of lymph node or distant metastasis, in differentiation types of gastric cancer classified by Lauren and Goseki and presence of vascular and neural infiltration.

It is known that enhanced production of reactive oxygen species and consequently oxidative stress may be engaged in cancer development via regulation of numerous signalling pathways including phosphoinositide-3-kinase (PI3K)/Akt-regulated signalling cascades, mitogen-activated protein (MAP) kinase/Erk cascade as well as the IκB kinase (IKK)/nuclear factor κB (NF-κB)-activating pathways ¹⁰. For example, the activation of the MAPK/Erk1/2 pathway in cancer leads to increased cell proliferation; PI3K/Akt activation affect on cell survival via phosphorylation and inactivation of its substrates – the pro-apoptotic proteins Bad, Bax, Bim or FOXO transcription factors leading to the inhibition of apoptosis whereas activation of NF-κB described as” redox-regulated sensor for oxidative stress” takes part in tumour cell survival, regulation of cell cycle and proliferation, cellular adhesion and migration and development of drug resistance ¹⁰. The role of H₂O₂ in carcinogenesis and activation of signalling pathways is particularly often emphasised because H₂O₂ may be a second messenger in cellular signalling. Its high concentration in the cell may induce DNA damages, inflammation (via the activation of NFκB) and changes in cellular metabolism ¹¹. Moreover, H₂O₂ can also damages membranes and other organelles, leading to the autophagy and mitophagy, and HIF1 activation ¹². Under conditions of oxidative stress the consequence of mitophagy is aerobic glycolysis and lactate production ¹³. One of the main enzymes responsible for H₂O₂ conversion from the superoxide radical is superoxide dismutase (SOD) considered as one of the most effective intracellular enzymatic antioxidants ¹⁴. In our work we observed significantly higher SOD activity in patients with gastric cancer in comparison with healthy control and the consequence of which may be enhanced generation of H₂O₂ in GC patients. Simultaneously, SOD increase may be an adaptive reaction to overproduction of ROS by gastric cancer cells ¹⁵. We also observed lower SOD activity in patients with tumours restricted to the mucosa and submucosa (T1) and muscularis propria (T2) in comparison with patients with tumours penetrating to the deeper layers – subserosa and serosa (T3) and adjacent structures (T4). It is clearly indicated that SOD activity increases with depth of tumour invasion. In our study SOD was considerably higher in patients with distant metastasis compared to those without metastasis. We may conclude that SOD may be enagaged in the metastasis process. Moreover, studies performed on cell lines confirmed that abnormally high SOD expression can regulate cellular proliferation and metastasis, possibly by promotion of cell cycle progression and inhibition of apoptosis ¹⁶. Additionally, elevated SOD levels enhance the invasive potential of cancer cells through suppressing cell proliferation. Moreover, lack of balance between generation of superoxide radical and H₂O₂ degradation in cells with high level of SOD may induce the metastatic potential of cancer cells ¹⁷. The effect of overproduction of ROS including H₂O₂ is counterbalanced by the activity of antioxidant enzymes like CAT, GPx and GR. One of the major enzymes responsible for H₂O₂ detoxification and its convertion to water and oxygen is catalase. One molecule of catalase can neutralize over 6 million molecules of H₂O₂ per minute and therefore it is considered as one of the most catalytically active enzymes ¹⁸. In this research work, the activity of catalase was significantly lower in patients with gastric cancer in comparison with the control group. It may be conclude that decreased CAT level predisposes the development of cancer as a result of increased levels of H₂O₂¹⁹. This theory is confirmed by studies carried out on mice in which it was demonstrated that at 9 months of age, female catalase-deficient mice spontaneously develop mammary tumors, which can be prevented by addition of vitamin E to their diet ²⁰. Japanese pre-clinical studies have demonstrated that therapy with catalase (via intravenous and intraperitoneal injection of catalase) in rodent animal models is sufficient to almost completely prevent tumour recurrence and metastasis ^21,22. Interestingly, we observed higher CAT activity in patients in T1 + T2 stages in comparison with T3 + T4, in patients with intestinal type of gastric cancer compared to diffuse type and in I + II types of gastric cancer according to Goseki classification than in III + IV. The differences in CAT activity occured in groups of patients divided according to histological features clearly indicated that depth of tumour invasion as well histological structure of the tumour showed a strong association with CAT activity and consequently H₂O₂ detoxification. Intestinal type of gastric cancer is defined by tumour cells arranged in tubular or glandular formations and is often linked to intestinal metaplasia. Moreover, this type of GC is associated with lymphatic or vascular invasion and exhibits a longer course and better prognosis ²³. Diffuse type arises from the gastric mucosa and is linked to gastritis and the tumour cells lack adhesion and infiltrate the stroma as single cells or small groups. Intracellular mucus can shift the nucleus of the cell aside to form signet-ring cell carcinoma. This type of cancer generally occurs in the whole stomach and is characterized by shorter duration and worse prognosis than intestinal type ²⁴. Thus, CAT activity seems to be strongly associated with histological structure of the tumour and its lower activity observed in diffuse type may be a factor indicating for worse prognosis for GC patients. Goseki histological classification is established on two cancer features: degree of tubular differentiation and the degree of mucus production by cancer cells ²⁵. Therefore, differences in CAT activity between patients with I + II types and III + IV may indicate that degree of tubular differentiation and mucus production is associated with detoxification of hydrogen peroxide by catalase. The other basic components of antioxidant barrier are glutathione peroxidase, glutathione reductase and non-enzymatic antioxidant – reduced glutathione. Glutathione metabolism is a key element of antioxidative defence mechanisms and protects cells from oxidative stress by reducing disulfide bonds of cytoplasmic proteins to cysteines and is oxidized to glutathione disulfide (GSSG) ²⁶. Maintaining the redox state of critical protein sulfhydryls are crucial for DNA repair. GPx catalyze the breakdown of H₂O₂ to H₂O and lipid hydroperoxides (ROOH) to alcoholes whereas GR reduces GSSG and refills GSH pools ²⁷. GPx participates in conversion H₂O₂ at low ROS level unlike catalase which is involved in H₂O₂ conversion at high ROS level ²⁸. It may be explained by the fact that GPx reveals a much higher affinity to H₂O₂ probably due to the different values of Michaelis-Menten constant (Km) of CAT and GPx. The Km for GPx (Km = 1 x 10^− 6 M) is considerably lower that the Km for CAT (Km = 2.4 x 10^− 4 M) which suggests that CAT scavenges H₂O₂ efficiently at higher concentrations ²⁹. We observed that activity of GPx, GR and concentration of non-enzymatic antioxidant – GSH was decrease in patients with gastric cancer. Decrease of all components of the antioxidant barrier (GPx, GR and GSH) in GC patients clearly shows exhaustion of antioxidant reserves in GC patients and predisposition them to redox imbalance as well as oxidative damages ⁸. GPx activity like CAT was statistically siginificantly increased in patients in T1 + T2 stages in comparison with T3 + T4. GR and GSH activity was increased in patients without lymph node metastasis compared to those with metastasis whereas GSH was also higher in patients without distant metastasis in comparison with metastasis. GPx, GR and GSH level was considerably higher in patients with intestinal type of gastric cancer according to Lauren’s classification compared to diffuse type and in patients without vascular infiltration. We demonstrated significantly increase of GSH activity in patients with I + II types of gastric cancer according to Goseki classification than in III + IV and in patients without neural invasion. Our observations clearly indicate that depletion of the antioxidant barrier manifested by a decrease in enzyme activity intensifies with depth of tumour invasion, development of metastasis and vascular and neural invasion as well as presence of more aggressive and worse prognosis type of gastric cancer according to Lauren and Goseki classification.

Evaluating the redox status on the basis of the level of single antioxidants may be difficult, hence we assessed the biomarkers reflecting resultant antioxidant/oxidant capacity (TAC, TOS, OSI) in patients with gastric cancer. TAC involve the cumulative effect of all antioxidants (both enzymatic and non-enzymatic), whereas TOS enable for the evaluation of all oxidants present in a blood sample ³⁰. OSI (TOS/TAC ratio) reflects the ratio between pro-oxidants and antioxidants. In our work we observed significantly lower TAC level and higher TOS and OSI value in patients with gastric cancer in comparison with healthy people. These results indicate on weakened antioxidant barier caused by overproduction of free radical production in patients with gastric cancer. Moreover, high OSI level indicates that oxidation processes outbalance the antioxidant protection. Additionally, shift of redox balance to the oxidation side resulting in higher sensitivity to oxidative stress and greater tendency to oxidative damages development. It is not suprisingly that we found higher TOS level in patients with poorly differentiated tumours (G3) than in moderately differentiated (G2). TOS and OSI were also increased in patients tumours penetrating into deeper layers (T3 and T4) in stomach as well as in patients with lymph node metastasis. TOS was considerably increased in patients with distant metastasis. These observartions suggest that oxidative stress seems to be a key driver of the malignant transformation observed in primary tumors and our observations indicate that it may enhances their metastatic potential. Piskounova et al. demonstrated that increased ROS production is essential to enable and sustain a highly metastatic phenotype of cancer ³¹.

Oxidation processess are essential for aerobic life, but ROS overproduction is harmful and their insufficient removing by antioxidant defense systems leads to proteins and DNA oxidation and lipid peroxidation that may take part in the multistage carcinogenesis process. Proteins are the main point of action of ROS in biological systems because of their abundance and high rate constants for reaction. Modification in proteins structure occured as a consequence of ROS action may include oxidation of the side chains of almost type of aminoacids, chain fragmentation, cross-linked protein aggregates and disorder of the secondary and tertiary structure ³². The most common assessed markers of protein oxidation are total thiols, ischemia-modified albumin (IMA) and advanced oxidation protein products (AOPP). Thiols belong to the group of organic compounds containing the sulfhydryl groups and participate in the control of antioxidant defence mechanisms. Thiols are considered as powerful antioxidants because they may be an electron acceptors via reducing unstable free radicals ³³. We showed a decreased concentration of total thiols in GC patients compared to healthy control. Moreover, total thiols level was increased in poorly differentiated tumours (G3), in patients with lymph node metastasis and in III and IV types of GC according to Goseki classification. It has been observed that abnormalities in thiol/disulphide homeostasis may play a role in gastric carcinogenesis, and a higher level of oxidative stress may cause advanced and more aggressive course of disease ³⁴. We also found higher level of IMA and AOPP in GC patients. IMA is considered as a marker of ischemia and oxidative stress resulting from tissue hypoxia. The scientists describing IMA also as a marker of inflammation because it is well known that ischemia inititates a proinflammatory reactions leading to ROS production and consequently oxidative stress which may also induce malignant transformation ³⁵. AOPP are dityrosine containing crosslinked protein products which are generated mainly by chlorinated oxidants – hypochloric acid and chloramines arising from myeloperoxidase activity ³⁶. Moreover, AOPPs induce oxidative ignition of neutrophils, monocytes, and T lymphocytes, leads to the highly up regulation of dendritic cells and activate NfkB signalling pathway which enhances the generation of pro-inflammatory cytokines, but also increases free radical formation ⁸. In this work AOPP concentration was significantly higher in patients with a tumour diameter > 5cm, in tumours penetrating to the subserosa and serosa (T3) and adjacent structures (T4), in patients with adenocarcinoma mucinosum, with lymph node and distant metastasis as well as in patients with diffuse type of GC. It is not surprising that AOPP increased with increase of tumour diameter and with worse prognosing types of GC because oxidized proteins tend to form aggregates resistant to degradation by proteolytic enzymes and their accumulation in cells may result in a gradual loss of structure and function impairment. It confirms that abnormalities in protein breakdown processes are engaged in cancer progression ³⁷.

Uncontrolled ROS generation also results in lipid peroxidation and DNA oxidation. The process of lipid peroxidation may cause decreased fluidity and inreased permeability of membrane, as well as damages to membrane proteins but also is responsible for production a complex range of reactive carbonyl compounds, ketones and alkanes ³⁸. The main products of lipid peroxidation are lipid hydroperoxides formed as a result of fatty acid oxidation. At physiological levels LOOH show beneficial effects: they increase in a cellular adaptive responses and increase tolerance against oxidative stress via modulation of antioxidants. On the other hand, their uncontrolled production finally causes the initiation and execution of ferroptosis – iron-dependent programed cell death caused by the presence of oxidative stress and lipid peroxidation ³⁹. Ferroptosis take part in cellular proliferation, senescence, and differentiation. LOOH are considered as more stable compounds than free radicals and their determination in the serum could be useful to predict the oxidative stress in tissues ⁴⁰. In our work we found an increased level of LOOH in patients with gastric cancer. However, these markers are of limited value as they lack sensitivity and or specificity therefore we also assessed other marker of oxidative stress – 8-Iso-P which are more stable and specific products of lipid peroxidation product than LOOH. 8-Iso-P is a prostaglandin included in the F2 isoprostane class that is generated in vivo by the free radical-catalyzed peroxidation of arachidonic acid and cell membrane phospholipids ⁴¹. 8‑isoprostane is considered as a gold standard of oxidant injury and is formed when the endogenous antioxidants are exhausted hence it is not surprising that we showed significantly higher level of this compound in GC patients because level on enzymatic and non-enzymatic antioxidants was also decreased in our research. So far, the role of 8-Iso-P in carinogenesis has not been defined but it is postulated that it may be a risk factor of cancers ⁴². Lipid peroxidation is strongly associated with DNA damages because this process is responsible for generation aldehydes as secondary products, such as trans-4-hydroxy-2-nonenal (HNE), or malondialdehyde (MDA) which may result in the production of promutagenic oxidatively generated and exocyclic DNA adducts in human cells. But the main source of oxidative damages of DNA are ROS and include breakage of strands, DNA base alterations, DNA-protein crosslinks (the single and double-strand), exchange of genes between two identical sister chromatids, the subsequent formation of clastogenic factors, and modifications in the nitrogenous base structures, impairment of tumour suppressor genes and protooncogenes leading to the transformation of normal cells into cancer cells ⁴³. We showed increased level of DNA/RNA products in our research which confirm enahnced oxidation of DNA/RNa in patients with gastric cancer.

Summarising, GC development is strongly linked to an imbalance between enhanced oxidants production and antioxidants and consequently increased oxidative damage to proteins, lipids and DNA/RNA. We observed a decrease of activity of enzymatic (CAT, GPx, GR) as wll as non-enzymatic (GSH) antioxidants and a decrease of total antioxidant capacity in patients with gastric cancer. Moreover, TOS and OSI values were increased in GC patients. It is well known that a weakened antioxidant barrier is ineffective in ROS removing and resulting in protein, lipids and DNA damages and we observed in our research increased level of protein oxidation products (IMA, AOPP), lipid peroxidation products (LOOH, 8-Iso-P) as well as DNA/RNA products. We also observed differences in oxidative stress parameters between groups of GC patients divided according to histopathological parameters like tumour size, histological type of the tumour, histological differentiation grade, tumour invasion depth, presence of lymph node and distant metastasis, Lauren and Goseki classification and presence of vascular and neural infiltration. Many redox biomarkers allow to distinguish patients with gastric cancer from healthy people with high sensitivity and specificity. Therefore, our study is an initial step for further clinical trials concentrating on the evaluation of the diagnostic utility of redox biomarkers in a larger population of patients with gastric cancer. Determination of redox parameters may be useful in the assessment of the tumour progression. Evaluation of the link between the intensity of oxidative stress and the survival rate of patients with gastric cancer also seems appropriate.

Finally, some of limitations of our research are also worth considering. Our research has been conducted on a small group of patients; hence, further research should be carried out on a greater group of patients with gastric cancer. The level of redox biomarkers was determined only in blood samples (serum/plasma), giving our results an approximate value. The redox parameters assessed in this paper are not specific to GC patients, therefore should be evaluated after the exclusion of other oxidative stress-associated diseases. However, the great advantage of our research is a carefully selected group of gastric cancer patients and control group without any coexistence diseases and the fact that some of determined redox parameters may be applied in non-invasive GC diagnostics. Our study may be a pre-eliminary step for further clinical trials evaluating the redox biomarkers and their diagnostic utility in a larger population of patients with gastric cancer.

Patients

The study was conducted on a group of 50 patients diagnosed with gastric cancer, who

were treated surgically in the 2nd Clinical Department of General and Gastroenterological Surgery Unit at the Medical University of Bialystok Clinical Hospital in 2017–2020. Patients were included in the study group based on a diagnosed adenocarcinoma at any stage without coexistence of other systemic diseases who did not receive radio- or chemotherapy before the surgery. The exclusion criteria were followed: squamous cell carcinoma and other non epithelial neoplasms, metastases of other neoplasms to the stomach, acute inflammatory diseases, infectious and autoimmune diseases (HIV/AIDS, hepatitis, Crohn’s and Hashimoto’s disease, ulcerative colitis, rheumatoid arthritis and psoriasis), cardiovascular diseases, metabolic diseases, such as osteoporosis, type 1 diabetes, mucopolysaccharidosis and gout, digestive, respiratory or genitourinary systems diseases. Additionally, smokers and patients taking other drugs (antibiotics, non-steroidal anti-inflammatory drugs, glucocorticosteroids, vitamins, and dietary supplements) for the three months before the surgery were excluded from the study. Lack of complete medical documentation was also an exclusion criterion.

The time from cancer diagnosis to the surgical resection of the tumour ranged from 2 days to 4 weeks. The study material was obtained from all patients before surgery.

The control group consisted of 50 healthy patients (selected by sex and age to match the study group) who participated in the follow-up study from January 2018 to January 2020. To the control group were qualified patients with normal results of complete blood count (CBC) and biochemical blood tests (Na+, K+, creatinine, AST, ALT).

The number of patients in the study and in the control groups was set based on a previously conducted pilot study. The study power was set at 0.8.

The study was conducted according to the guidelines of the Declaration of Helsinki and the protocol was approved by the Bioethics Human Research Committee of the Medical University of Bialystok (Permission No. APK-002/238/2022). All subjects gave their informed consent for inclusion before they participated in the study.

Histopathological analysis

In a routine histopathological analysis we assessed the histological type of tumour, the grade of histological malignancy according to the World Health Organization (WHO) guidelines ⁴⁴, tumour stage according to the TNM classification standard of the Union for International Cancer Control ⁴⁵ including depth of tumour invasion (pT), presence of lymph node metastasis (pN) and distant metastasis (pM), tumour type according to Lauren’s ⁴⁶ and Goseki ²⁵ classification, vascular and neural infiltration and degree of desmoplasia.

Tissue sections taken during the surgery were fixed in 4% buffered formalin solution and embedded in paraffin at a temperature of 56°C. The paraffin blocks were then sliced with a microtome (Microm H340) into approximately 4 µm-thick slides and stained with hematoxylin and eosin. The obtained sections were reviewed by two independent pathologists on a microscope Olympus CX22 under 200× and 400× magnification.

Sample Collection and Storage

Fasting venous blood (10 ml) was taken from all patients on an empty stomach, upon overnight rest, using the S-Monovette® K3 EDTA and S-Monovette® serum collection system (Sarstedt, Germany). Immediately after collection, blood was centrifuged at 1500 x g for 10 min at + 4°C (MPW 351, MPW Med. Instruments, Warsaw, Poland) to separate plasma or serum from erythrocytes. The top layer (plasma or serum) was then taken. 0.5 M butylated hydroxytoluene (20 µl/2 ml plasma or serum) was added to prevent sample oxidation. Until redox determinations, all samples were stored at -80°C.

Redox assays

All reagents (unless otherwise stated) were of analytical grade and were purchased from Sigma-Aldrich Saint Louis (MO, USA) or Sigma-Aldrich Nümbrecht (Germany).

Infinite M200 PRO Multimode Microplate Reader (Tecan Group Ltd., Männedorf, Switzerland) was used to measure absorbance/fluorescence.

The fluorescence was assessed in 96-well black-bottom microplates. All assays were performed in duplicate samples. The results were standardised to 1 mg of total protein. According to the manufacturer's instructions, total protein content was measured spectrophotometrically (Thermo Scientific PIERCE BCA Protein Assay; Rockford, IL, USA).

Antioxidant enzymes and non-enzymatic antioxidants

CAT (EC 1.11.1.6) activity was measured by the colorimetric method, measuring the decomposition rate of hydrogen peroxide (H₂O₂) at a wavelength of 240 nm ⁴⁷. A unit of CAT activity was defined as the amount of the enzyme catalysing the decomposition of 1 mmol H₂O₂ per 1 minute.

GPx (EC 1.11.1.9) activity was determined colorimetrically by measuring the oxidation of NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) at a wavelength of 340 nm ⁴⁸. A unit of GPx activity was defined as the amount of enzyme that catalyses the oxidation reaction of 1 mmol NADPH per 1 minute.

GR activity (EC 1.8.1.7) was determined using the colorimetric method by measuring the decrease in NADPH absorbance at a wavelength of 340 nm ⁴⁹. A unit of GR activity was defined as the amount of enzyme catalysing the oxidation of 1 µmol NADPH in a 1 minute.

SOD-1 (E.C. 1.15.1.1) activity was evaluated using colorimetric method by measuring the inhibition of adrenaline oxidation at a wavelength of 480 nm ⁵⁰. A unit of SOD activity was defined as the amount of the enzyme inhibiting adrenaline oxidation by 50%.

Glutathione concentration was determined colorimetrically using the enzymatic reaction with 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB), NADPH, and GR at a wavelength of 412 nm ⁵¹.

Redox status

The determination of the TAC level was performed using the colorimetric method by measuring changes in ABTS·+ (2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonate) absorbance at a wavelength of 660 nm ⁵².

TOS level was evaluated colorimetrically by measuring the oxidation of ferrous ion to ferric ion in the presence of oxidants in a serum sample ⁵³.

OSI calculation was the result of the formula: OSI = [TOS]/[TAC] x 100% ⁵⁴.

Oxidative damage to proteins, lipids and nucleic acids

Ellman's assay was used to assess the concentration of total thiols ^55,56. The absorbance was measured at a wavelength of 593 nm, and the total thiols content was calculated from the calibration curve for reduced glutathione.

IMA concentration was assessed using spectrophotometric method based on the measurement of the exogenous cobalt (Co²⁺) binding facility of the human serum albumin (HSA) ⁵⁷. The absorbance was assessed at a wavelength of 470 nm.

The concentration of AOPP was determined using colorimetric method by measuring iodide ion oxidising capacity of the sample at a wavelength of 340 nm⁠ ⁵⁸. Serum samples were diluted in 0.02 M PBS, pH 7.4 (1:5, v/v).

LOOH level was assayed with spectrophotometric method with the FOX-2 test based on the reaction of Fe³⁺ ions with xylenol orange (XO) ^59,60.

8-iso-P concentration was determined with an ELISA test using a complete reagent kit (8-Isoprostane ELISA Kit, Cayman Chemicals, Ann Arbor, MI, USA). The assay range was from 0,8 to 500 pg/ml and the sensitivity was about 3 pg/ml.

Plasma oxidative DNA/RNA damage concentrations were determined using commercial high sensitivity ELISA kits (DNA/RNA oxidative damage ELISA Kit, Cayman Chemicals, Ann Arbor, MI, USA). The test detects all three oxidized guanine species; 8-hydroxy-2′-deoxyguanosine, 8-hydroxyguanosine, and 8-hydroxyguanine. The assay range was from 10.3 to 3,000 pg/ml and the sensitivity was about 30 pg/ml.

Statistical analysis

GraphPad Prism 9.0 (GraphPad Software, La Jolla, USA) was used for statistical analysis. The normality of distribution was determined using the Shapiro–Wilk test. For a normal distribution, the Student's t-test was used. In the case of the lack of normal distribution, the Mann-Whitney U test was used. The results were presented as median (minimum-maximum), and the value of p < 0.05 was considered statistically significant. Spearman correlation coefficient was used to assess the relationship between redox and clinicopathological parameters. The diagnostic utility of the redox biomarkers was determined using Receiver Operating Characteristic (ROC). AUC (area under the curve) and optimal cut-off values were determined for each parameter, ensuring high sensitivity and specificity.

Author Contributions: Conceptualization, J.D.; Methodology, J.D., M.M.; Software, J.D., M.M., P.Ż.; Validation, J.D., M.M., P.Ż.; Formal Analysis, J.D., M.M., A.P.; Investigation, J.D., M.M., A.Z.; Resources, B.K., A.P.; Data Curation, J.D., M.M.; Writing – Original Draft Preparation, J.D.; Writing – Review & Editing, J.D., M.M., A.Z., J.M-K.; Visualization, J.D., A.P.; Supervision, J.D., M.M., ; Project Administration, J.D., M.M.; Funding Acquisition, J.D., M.M., J.M-K.”.

Data Availability: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests: The authors declare that there is no conflict of interest regarding the publication of this article.

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No competing interests reported.

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Diagnostic significance and utility of circulating redox biomarkers in patients with gastric cancer

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Abstract

Figures

Introduction

Results

Clinical characteristics

Antioxidant Defence

Total Antioxidant/oxidant Status

Oxidative Damage Products

ROC Analysis

Correlations

Discussion

Patients And Methods

Patients

Histopathological analysis

Sample Collection and Storage

Redox assays

Antioxidant enzymes and non-enzymatic antioxidants

Redox status

Oxidative damage to proteins, lipids and nucleic acids

Statistical analysis

Declarations

References

Additional Declarations

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