Effect of drinking water on changes in haematological parameters
Basic haematological parameters (WBC/ DC) in different study groups at baseline, 8 and 14-months post baseline assessments are shown in Table 1.
Table 1 Changes in basic hematology (Total WBC, Neutrophils and Lymphocytes) at the baseline (generalized outcome n=30), 8- and 14-month post baseline (n=10 per each experimental group) (Mean ± SEM).
Study Groups
|
Assessment parameter
|
Time of the assessment
|
|
WBC
|
Neutrophils
|
Lymphocytes
|
|
|
15,800±591
|
18.41±0.86
|
80.70±0.83
|
Baseline
|
NTM
|
10,360±808
|
24±1.67
|
75±2
|
8-month post BL
|
|
9,467±217
|
22±0.87
|
76.5±6.7
|
14-month post BL
|
NTMB
|
10,838±843
|
23.3±0.7
|
75.2±2.1
|
8-month post BL
|
|
11,817±1116
|
32±2.3
|
67.16±2.5
|
14-month post BL
|
DD
|
14,670±606*
|
27±1.27*
|
72±0.89
|
8-month post BL
|
|
12,133±589
|
30±1.7
|
68.5±1.5
|
14-month post BL
|
BB
|
11,425±787
|
19±1.75
|
79.3±1.76*
|
8-month post BL
|
|
10,567±557
|
29 ±1.8
|
70.6±11
|
14-month post BL
|
HW
|
12,427±505
|
22.6±0.95
|
75.25±1.04
|
8-month post BL
|
|
9,967±803
|
24±1.3
|
74.3±1.4
|
14-month post BL
|
CO
|
10,270±705
|
19.4
|
73±1.23
|
8-month post BL
|
|
10,466±557
|
31.5±2.3
|
66.5±2.5
|
14-month post BL
|
Past baseline assessment n=(10) each experimental groups.
When comparing total leucocyte (WBC) counts obtained at 8- months post baseline with the baseline values, all groups had significantly reduced total WBC counts except the rats that ingested water from DD (p<0.05). A significant increase in the neutrophil counts were observed in all the groups except in the BB and CO groups. Further, a significant reduction in lymphocytes were also observed in all the experimental groups except BB (p<0.05).
Inter-group comparison at 8-month post baseline revealed that both total WBC and neutrophil counts were significantly high in DD compared to the remaining groups (p<0.05). Further, BB reported a significantly high lymphocyte counts at the same assessment point (Table. 1).
Comparison of total WBC counts at 14-month post baseline with baseline values revealed a significant reduction in the counts in groups NTM, BB, HW and CO. However, the reductions in groups DD and NTMB were comparatively less when compared to the remaining groups.
At the 14- month post baseline, all the experimental groups except HW and NTM had significantly high neutrophil counts when compared to the baseline (p<0.05). When compared with the other groups, HW and NTM had significantly lower neutrophil counts at 14- month post baseline (p<0.05). A significant reduction in lymphocytes was observed in NTMB, BB, DD, and CO groups at 14- month post baseline when compared to the baseline (p<0.05). Inter-group comparison of lymphocyte counts at 14-month post baseline showed that both NTM and HW groups had a significant elevation when compared to NTMB, DD and CO groups (p<0.05).
Effect of drinking water on the changes in CD4+ and CD8+ lymphocyte subsets
Changes in CD4+, CD8+ and serum cytokines; TNFα, IL-1β, and IL6 levels in different study groups at baseline, 8- and 14- months post baseline are shown in Table 2.
At baseline, the lowest CD4+ lymphocyte count was observed in the CO control group compared to the other groups (p < 0.05). At 14-month post baseline, a significantly high CD4+ lymphocyte count was observed in the NTM group compared to NTMB (p < 0.05), BB, HW, and CO (p < 0.01). Group DD had the second highest CD4+ lymphocyte count at 14- month post baseline and it was significantly higher than BB, HW and CO groups (p<0.05). No significant difference in CD4+ lymphocyte counts were observed between NTM and DD at 14- month post baseline (p>0.05).
Table 2. Changes in CD4+, CD8+ at the baseline, and 14-month post baseline, serum cytokines; TNFα, IL6 and IL-1ß at the baseline and 8-month post baseline in different experimental groups (Mean±SEM).
Study Group
|
Assessment Parameters
|
Time of Assessment
|
|
CD4+
|
CD8+
|
Serum IL-1ß
(pg/mL)
|
Serum IL6
(pg/mL)
|
Serum TNFα (pg/mL)
|
|
|
3496±290
|
1270±112
|
654.6±119
|
720.5±152
|
ND
|
Baseline
|
NTM
|
|
|
267.7±66.2
|
42.5±29.2*
|
610.37±144.7*
|
8-month BL
|
|
3788±90.2
|
2491±196
|
|
|
|
14-month BL
|
NTMB
|
|
|
492.12.±76.66*
|
0.0*
|
52.03±14.7
|
8-month BL
|
|
3059±319
|
1082±264
|
|
|
|
14-month BL
|
DD
|
|
|
82.28±22.12
|
135.0±41.3
|
88.3±28.32*
|
8-month BL
|
|
3420±108
|
1353±166
|
|
|
|
14-month BL
|
BB
|
|
|
242.34±31.96
|
159.5±32.14
|
93.06±18*
|
8-month BL
|
|
2671±233
|
874±121
|
|
|
|
14-month BL
|
HW
|
|
|
326.2±107.87
|
235.7 ±67
|
61.98±8.54
|
8-month BL
|
|
2748±287
|
1491±161
|
|
|
|
14-month BL
|
CO
|
|
|
610.37±144.7*
|
158.67±36.4
|
19.48±2.45
|
8-month BL
|
|
2177±212
|
1155±212
|
|
|
|
14-month BL
|
At baseline, rats that ingesting water from NTM had significantly higher CD8+ lymphocyte counts compared to the other groups (p< 0.001). No significant difference was observed between other groups at baseline. At 14-month post baseline, a significantly high CD8+ lymphocyte count was observed in the NTM group compared to NTMB, DD, BB, HW and CO groups (p< 0.01). Rats ingesting water from BB had significantly low CD8+ lymphocyte count when compared to rats who ingested water from NTM and HW.
Effect of drinking water on the changes in serum cytokines; TNFα, IL-1β, and IL-6.
Serum TNFα, IL-1β and IL-6 levels in all test groups along with controls were assessed at baseline and 8-months post baseline and the comparison was carried out between the two assessment points.
At baseline, both serum IL-1β and IL-6 were measurable in serum, whereas serum TNFα levels remained undetected in all groups. The cytokines fluctuated differently between the two assessment points and the difference was evident among some of the test groups at 8-month post baseline (Table 2).
There was an overall reduction in both serum IL-1β and IL-6 between the two assessment points in all the test groups. A significant reduction in IL-1β was observed in both DD and BB groups while IL-6 reduction was significant in both NTM and NTMB groups when compared to the remaining experimental groups.
Serum IL-1β levels 8-month post baseline showed significantly higher levels in the NTMB and CO groups in comparison to the remaining groups, thus indicating a lower reduction between the two assessment points (Table 2).
Serum TNFα levels in all the experimental groups were not detected at baseline. However, there was overall increase in the levels at 8-month post baseline, with both DD and BB groups showing a significant surge when compared to the remaining groups (p < 0.05).
Effect of drinking water on the changes in liver cytokines; TNFα, IL-1β, and IL-6
TNFα, IL-1β and IL-6 in all experimental groups were assessed at 14-month post baseline and are shown in Table 3. Both TNFα and IL-6 levels in the DD group were significantly elevated when compared to the remining groups. No significant difference was observed in IL-1β levels among these groups.
Effect of drinking water on the changes in serum enzymes; AST and ALT
Both AST and ALT levels at baseline were consistent in all the experimental groups with an average value of 87.33U/L ±20.06, and 33.51U/L±2.83, respectively. There was an early increase in both AST and ALT levels among rats in NTM group and such elevation was evident up to 8-month post baseline. However, this elevation in both AST and ALT levels was not maintained at the 14-month post baseline. In NTMB group, a similar pattern of AST and ALT fluctuation was also observed (Table 3).
Table 3 Changes in serum AST, ALT at the baseline, 8- and 14-month post baseline and hepatic cytokines; TNFα, IL6, IL-1β at the 14-month post baseline in different experimental groups (Mean±SEM).
Study Groups
|
Assessment Parameter
|
Time of the assessment
|
|
ALT (U/L)
|
AST (U/L)
|
IL1ß pg/mg
|
IL6 pg/mg
|
TNFα pg/mg
|
|
|
33.51 ± 2.83
|
87.33 ± 20.1
|
ND
|
ND
|
ND
|
Baseline
|
NTM
|
40.85±1
|
115.93±8
|
|
|
|
8-months BL
|
|
38.70±4.5
|
97.75±8
|
81.18±1.26
|
177.45±52.50
|
64.71±11.83
|
14-month BL
|
NTMB
|
44.86±2
|
214.41±9*
|
|
|
|
8-months BL
|
|
43.25±5
|
92.30±6
|
89.87±5.29
|
164.89±56
|
66.07±12.13
|
14-month BL
|
DD
|
43.60±1
|
152.80±7*
|
|
|
|
8-months BL
|
|
30.18±2
|
93.58±16
|
79.09±10.88
|
194.81±30.06
|
76.33±6.87
|
14-month BL
|
BB
|
43.78±3
|
87.09±4
|
|
|
|
8-months BL
|
|
36.73±1
|
92.40±2
|
84.35±4.44
|
140.07±45.09
|
55.67±13.48
|
14-month BL
|
HW
|
49.51±4
|
203.83±1*
|
|
|
|
8-months BL
|
|
123.5±3*
|
175,80±2*
|
87.50±20.59
|
149.55±58.04
|
28.11±9.16
|
14-month BL
|
CO
|
42.32±1
|
114.23±3
|
|
|
|
8-months BL
|
|
33.68±2
|
88.41
|
67.68±
|
147.12±53.43
|
61.58±8.43
|
14-month BL
|
Rats in DD group showed a significantly elevated AST levels at 8-month post baseline, but it was not maintained at 14-month.
Rats in the HW group showed a consistently elevated AST levels from 8-month post baseline until termination of the study at 14 months. Interestingly, ALT in the same group was also elevated at 14-month.
The remaining groups did not show any significant changes in both AST and ALT levels from baseline.
Effect of drinking water on the changes of liver and renal histopathology
The main histopathological change observed in renal tissues were peritubular non suppurative interstitial nephritis whilst in liver tissues, it was observed steatosis, portal tract and parenchymal inflammatory cell infiltrates as prominent lesions [11]. Non-affected to affected kidney tissue ratios are summarized in the Table 4 and the percentage severity of liver lesions are summarized in Table 5.
Table 4 Mean non-affected to affected renal microscopic field ratios of rats that ingested water from different sources.
Parameter
|
NTM
|
NTMB
|
BB
|
DD
|
HW
|
CO
|
Mean non affected/affected microscopic field ratios of the kidney tissues
|
4.55
|
3.233
|
2.98
|
3.322
|
4.85
|
4.546
|
Table 5 Percentages of different hepatic histopathological changes observed in rat that ingested water from different sources.
Experimental groups
|
Steatosis (%)
|
Inflammatory Lesions in the portal tract (%)
|
Inflammatory Lesions in the parenchyma (%)
|
Inflammatory Lesions in the parenchyma & portal tracts (%)
|
NTM
|
40%
|
80%
|
60%
|
60%
|
NTMB
|
33%
|
67%
|
67%
|
33%
|
DD
|
30%
|
90%
|
70%
|
67%
|
BB
|
33%
|
78%
|
56%
|
44%
|
HW
|
60%
|
60%
|
30%
|
20%
|
CO
|
50%
|
25%
|
37.5%
|
0%
|