Cyclophosphamide- and doxorubicin-induced impairment of high affinity choline uptake and spatial memory can be prevented by dietary choline supplementation in breast tumor bearing mice – PubMed Black Hawk Supplements

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AC chemotherapy (Adriamycin and Cytoxan, i.e., doxorubicin and cyclophosphamide, respectively), a common treatment for breast cancer, can lead to significant cognitive side effects, known as Chemotherapy-Related Cognitive Impairments (CRCIs). These cognitive impairments can persist over 20 years and significantly affect the quality of life for cancer patients and survivors. AC chemotherapy is known to impair ovarian function and reduce circulating estradiol (E2), an effect that can decrease…
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Cyclophosphamide- and doxorubicin-induced impairment of high affinity choline uptake and spatial memory can be prevented by dietary choline supplementation in breast tumor bearing mice - PubMed

Cyclophosphamide- and doxorubicin-induced impairment of high affinity choline uptake and spatial memory can be prevented by dietary choline supplementation in breast tumor bearing mice

Robert Botelho et al. PLoS One. .

Abstract

AC chemotherapy (Adriamycin and Cytoxan, i.e., doxorubicin and cyclophosphamide, respectively), a common treatment for breast cancer, can lead to significant cognitive side effects, known as Chemotherapy-Related Cognitive Impairments (CRCIs). These cognitive impairments can persist over 20 years and significantly affect the quality of life for cancer patients and survivors. AC chemotherapy is known to impair ovarian function and reduce circulating estradiol (E2), an effect that can decrease high-affinity choline uptake (HACU) and reduce acetylcholine (ACh) availability. Because ACh is involved in attention, learning and memory function we hypothesized that the cognitive deficits observed during and after adjuvant chemotherapy (AC) are associated with compromised high affinity choline uptake (HACU) due to suppressed ovarian function. Increasing available choline has been demonstrated to enhance HACU under conditions of demand for ACh, therefore we propose that choline supplementation can mitigate CRCIs by maintaining cholinergic function throughout and following chemotherapy treatment. Our study demonstrates cognitive deficits in tumor-bearing but not non-tumor-bearing mice during and following AC chemotherapy, suggesting that tumors enhance vulnerability to CRCIs. We found that HACU was impaired in tumor-bearing mice administered AC chemotherapy and that a choline-enriched diet can mitigate both the reduction of HACU induced by chemotherapy and deficits in spatial memory, suggesting a protective role of dietary choline against disruptions in HACU and cognitive impairment caused by chemotherapy. This underscores the potential use of dietary choline supplementation as a part of chemotherapeutic interventions.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Study timeline and methodological overview.

Female mice were monitored weekly for tumor development through palpation and external measurement. Day 1 indicates the first day of the week of tumor identification. Following baseline MWM testing, mice were assigned to groups on Day 8, followed by orientation and training in the Morris Water Maze (MWM) from Days 11 to 18 to evaluate spatial memory. Following baseline MWM testing Mice either continued on a standard diet or transitioned to a 2% choline diet on Day 18. Assessment of proestrus status and submandibular blood draws were conducted on Days 15–18, 43–46, and 71–74. Mice received weekly injections of either a combination of cyclophosphamide (CYP; 66.7 mg/kg, i.v.) and doxorubicin (DOX; 6.7 mg/kg, i.v.) or saline on Days 19, 26, 33, and 40. Subsequent evaluations of spatial memory were conducted on Days 46 and 74 to assess the impact of chemotherapy. Mice were euthanized via decapitation and tissue was collected for high-affinity choline uptake (HACU) assays between Days 22–24, 50–52, and 78–80.

Fig 2
Fig 2. Longitudinal assessment of animal weights and tumor metrics.

For panels A-D, values represent group means +/- S.E.M.; the shaded red area indicates week 1 through week 4 of cyclophosphamide (CYP; 66.7 mg/kg, i.v.) and doxorubicin (DOX; 6.7 mg/kg, i.v.) or saline injections; Color and symbol references for groups are in the legend. Initial group sizes for non-tumor bearing mice: standard diet saline injected (n = 81); standard diet CYP+DOX- injected (n = 69); 2% choline diet saline injected (n = 54); 2% choline diet CYP+DOX- injected (n = 49). Initial group sizes for tumor bearing mice: standard diet saline injected (n = 58); standard diet CYP+DOX-injected (n = 49); 2% choline diet saline injected (n = 64); 2% choline diet CYP+DOX-injected (n = 53). For all comparisons, a p0.05 after Sidak’s correction for family-wise error was considered significant. A) Weights of mice in each experimental group throughout the study. This panel illustrates the impact of 2% choline, introduced on Day 18, and weekly injections on Days 19, 26, 33, and 40 of CYP+DOX on weight. B) Number of detectable tumors in tumor-bearing mice, reflecting tumor incidence and proliferation under different treatment conditions. C) Total tumor volume of mice, reflecting the summed volumes of all tumors per mouse to demonstrate the growth dynamics of tumors in response to the experimental interventions. D) Total tumor volume normalized by animal weight to account for the influence of body mass on tumor size.

Fig 3
Fig 3. Frequency of proestrus in non-tumor and tumor-bearing female mice.

The percentage of female mice exhibiting proestrus [an impedance measurement of ≥ 4 kΩ on at least 1 of the 4 consecutive days of measurement] at baseline (15–18 days), four weeks of chemotherapy (43–46 days), and post-chemotherapy (71–74 days). Initial group sizes for non-tumor bearing mice: standard diet saline injected (n = 81); standard diet CYP+DOX- injected (n = 69); 2% choline diet saline injected (n = 54); 2% choline diet CYP+DOX- injected (n = 49). Initial group sizes for tumor bearing mice: standard diet saline injected (n = 58); standard diet CYP+DOX-injected (n = 49); 2% choline diet saline injected (n = 64); 2% choline diet CYP+DOX-injected (n = 53). The shaded red area indicates week 1 through week 4 of cyclophosphamide (CYP; 66.7 mg/kg, i.v.) and doxorubicin (DOX; 6.7 mg/kg, i.v.) or saline injections. Color and symbol references for groups are in the legend. Arrows indicate significant changes from the baseline within each group, where asterisks denote significant differences between groups at the same timepoint. For all comparisons, a p0.05 after Sidak’s correction for family-wise error was considered significant.

Fig 4
Fig 4. Spatial exploration patterns of non-tumor bearing mice under different treatment conditions in the Morris Water Maze (MWM).

The proportion of distance moved for non-tumor bearing mice exploring each of six designated zones in the MWM during the probe trial. Bars represent group means +/- S.E.M.; Dashed line represents chance or unbiased performance (16.67%); 0° corresponds to the target zone where the platform was located during training and platform trials. Each row represents one of the four experimental groups—based on diet (standard or 2% choline) and treatment (saline or CYP+DOX)—across three key time points: baseline (Day 18), after four weeks of treatment (Day 46), and post-chemotherapy (Day 74). Group sizes for standard diet saline injected mice (Day 18 n = 81; Day 46 n = 61; Day 74 n = 48); standard diet CYP+DOX- injected mice (Day 18 n = 69; Day 46 n = 50; Day 74 n = 38); 2% choline diet saline injected mice (Day 18 n = 54; Day 46 n = 33; Day 74 n = 21); 2% choline diet CYP+DOX- injected mice (Day 18 n = 49; Day 46 n = 37; Day 74 n = 27). Upward arrows indicate a greater increase in exploration of a given zone relative to chance, suggesting targeted search behavior, while downward arrows depict less of the zone than chance/unbiased performance, suggesting avoidance or random search patterns. Asterisks denote statistical significance. For all comparisons, a p0.05 after Sidak’s correction for family-wise error was considered significant.

Fig 5
Fig 5. Spatial exploration patterns of tumor bearing mice under different treatment conditions in the Morris Water Maze (MWM).

The proportion of distance moved for tumor-bearing mice exploring each of the six designated zones in the MWM during the probe trial. Bars represent group means +/- S.E.M.; Dashed line represents chance or unbiased performance (16.67%); 0° corresponds to the target zone where the platform was located during training and platform trials. Each row represents one of the four experimental groups—based on diet (standard or 2% choline) and treatment (saline or CYP+DOX)—across three key time points: baseline (Day 18), after four weeks of treatment (Day 46), and post-chemotherapy (Day 74). Group sizes for standard diet saline injected mice (Day 18 n = 58; Day 46 n = 45; Day 74 n = 20); standard diet CYP+DOX-injected mice (Day 18 n = 49; Day 46 n = 36; Day 74 n = 23); 2% choline diet saline injected mice (Day 18 n = 64; Day 46 n = 45; Day 74 n = 19); 2% choline diet CYP+DOX-injected mice (Day 18 n = 53; Day 46 n = 40; Day 74 n = 20). 2 Upward arrows indicate greater exploration of a given zone relative to chance, suggesting targeted search behavior, while downward arrows depict less exploration of the zone than chance/unbiased performance suggesting avoidance or random search patterns. Asterisks denote statistical significance. For all comparisons, a p0.05 after Sidak’s correction for family-wise error was considered significant.

Fig 6
Fig 6. High-affinity choline Uptake (HACU) in the Cortex, Striatum, and Hippocampus of non-tumor and tumor-bearing mice.

HACU rates (pmol/mg protein/min) in the frontal cortex, striatum, and hippocampus of non-tumor bearing and tumor-bearing mice. Bars represent group means +/- S.E.M. Each row illustrates HACU in a brain region at different post-injection intervals: 3–5 days following one injection (Day 22–24), 10–12 days following four injections (Day 55–52), and 35–37 days after the last of four injections (Day 78–80). Group sizes for non-tumor bearing mice: standard diet saline injected (Days 22–24 n = 12; Day 55–52 n = 12; Day 78–80 n = 12); standard diet CYP+DOX- injected (Days 22–24 n = 12; Day 55–52 n = 12; Day 78–80 n = 12); 2% choline diet saline injected (Days 22–24 n = 12; Day 55–52 n = 9; Day 78–80 n = 12); 2% choline diet CYP+DOX- injected (Days 22–24 n = 12; Day 55–52 n = 10; Day 78–80 n = 12). Initial group sizes for tumor bearing mice: standard diet saline injected (Days 22–24 n = 12; Day 55–52 n = 11; Day 78–80 n = 9); standard diet CYP+DOX-injected (Days 22–24 n = 12; Day 55–52 n = 9; Day 78–80 n = 12); 2% choline diet saline injected (Days 22–24 n = 12; Day 55–52 n = 12; Day 78–80 n = 12); 2% choline diet CYP+DOX-injected (Days 22–24 n = 12; Day 55–52 n = 12; Day 78–80 n = 12). Treatment groups are compared within each brain region. Asterisks denote significant differences in HACU rates. For all comparisons, a p0.05 after Sidak’s correction for family-wise error was considered significant.

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Cyclophosphamide- and doxorubicin-induced impairment of high affinity choline uptake and spatial memory can be prevented by dietary choline supplementation in breast tumor bearing mice – PubMed