Mol. Cells 2015; 38(3): 236-242
Published online January 16, 2015
https://doi.org/10.14348/molcells.2015.2282
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: yshim@konkuk.ac.kr
Intake of caffeine during pregnancy can cause retardation of fetal development. Although the significant influence of caffeine on animal development is widely recognized, much remains unknown about its mode of action because of its pleiotropic effects on living organisms. In the present study, by using
Keywords caffeine,
1,3,7-trimethylxanthine, commonly known as caffeine, is one of the most popular drugs in the world. It comes from coffee beans, tea leaves, kola nuts, cacao pods, and so on (Gilbert et al., 1976). People ingest caffeine in one form or another every day, and it has become very popular in our daily lives. Recently, caffeine consumption has been increasing even among teenagers. Caffeine is known to enhance physiological functions in humans including sharpening our mind, improving athletic performance, and many other health improvements (Reviewed in Weinberg and Bealer, 2002). However, caffeine is an addictive drug. Although its effects are milder than those of other addictive drugs such as amphetamines, cocaine, and heroin, overlapping channels mediate the signal (Nehlig, 1999), which means that caffeine can also cause medical concerns.
Research revealed that caffeine consumption both before and during pregnancy increased the risk of spontaneous abortion (Cnattingius et al., 2000) and births classified as ‘small for gestational age’ (Hoyt et al., 2014). Maternal caffeine intake of over 300 mg/day doubled the risk of miscarriage compared with that of 151 mg/day (Giannelli et al., 2003). Such results suggest that intake of high doses of caffeine during pregnancy is a risk factor for fetal growth retardation. Therefore, understanding the basis of developmental defects caused by caffeine intake is an emerging issue, considering the fact that caffeine is now intertwined in the daily life of most people. Caffeine is metabolized in the liver by cytochrome P450 (CYP450) and excreted as urine in mammals (Kot and Daniel, 2008). The half-life of caffeine ranges from 3 to 7 h in adult plasma. Interestingly, the half-life is prolonged during pregnancy, in which it can be more than 10 h (Knutti et al., 1981). In addition, neonates have a greatly reduced capacity to metabolize caffeine, and it is excreted into the urine in largely unmetabolized form until hepatic metabolism becomes activated (Brent et al., 2011). Mammals contain 51 CYP family members, which are grouped into 10 subfamilies (Nelson et al., 2004). Among them, CYP1 to CYP4 are the drug-metabolizing families, and caffeine has been shown to be metabolized by CYP1A2 in mammals (Kot and Daniel, 2008). CYP450s render drugs more water-soluble so they can be excreted more easily in the urine or bile, and thereby, the drugs are processed by oxidative metabolism. Thus, CYP450s are able to promptly reduce the effects of drugs. The reduced capacity of neonates to metabolize caffeine therefore suggests that insufficient neonatal
Methods for the maintenance and handling of
L4-stage N2 hermaphrodites were individually cloned onto either caffeine-containing or control NGM agar plates and grown at 20°C. They were transferred to new plates in 24 h intervals for three days to allow embryo production. Laid embryos were considered dead if they did not hatch after 48 h at 20°C. Brood size was calculated as the total number of non-hatched and hatched embryos produced by a single mother hermaphrodite. Embryonic lethality was calculated as the percentage of non-hatched embryos among the total number of embryos produced. Percent larval development was calculated as the percentage of larvae that reached the adult stage among the total number of hatched embryos, as previously described (Kawasaki et al., 2013). The broods of 10 mother hermaphrodites were examined to calculate the above values at each concentration of caffeine treatment.
Synchronized L1-stage worms were incubated on the plate containing 0 or 30 mM caffeine in the absence of food,
Synchronized L1-stage worms were grown on NGM agar plates with or without caffeine for 3 days. Total RNA was extracted from the worms with Trizol reagent (Sigma, USA) and purified, after which the first strand cDNA was synthesized by M-MLV reverse transcriptase (Gibco BRL, USA) using oligo-dT primer (Promega, USA).
Synchronized L1-stage or L4-stage worms were grown on NGM agar plates with or without caffeine for 24 h. Total RNA was extracted from the worms with Trizol reagent (Sigma, USA), purified, and reverse transcribed with M-MLV reverse transcriptase (Gibco BRL, USA) using oligo-dT primer (Promega, USA) to synthesize the first strand cDNA. Respective cDNA products were PCR-amplified using the following primers:
RNAi analysis was performed using the “RNAi-by-soaking” method, as described previously (Maeda et al., 2001). N2 worms were synchronized at the L1 larval stage in the absence of food, after which they were soaked for 24 h in each of the double-strand RNA solutions transcribed
All experiments were repeated more than three times for statistical evaluation of data.
As a preliminary study, we examined growth rates of worms after treatment of 0, 5, 10, 15, 20, and 30 mM caffeine. We found that effects of caffeine were dosage-dependent, and determined three different concentrations of caffeine, 10, 20, and 30 mM as mild, moderate, and critical doses, respectively. To examine the effects of caffeine on the developmental process of
The effect of caffeine on the larval development of
To confirm that the larvae arrested with the 30 mM caffeine treatment indeed did not reach an adult stage, the expression of
Next, the effect of caffeine during different stages of larval development was examined by treating the worms with 30 mM caffeine starting from the L1, L2, L3, L4, or young adult stage (Fig. 3). Worms treated with 30 mM caffeine since the L1 stage were found to be mostly arrested at the L1 larval stage, as observed above. In contrast, the majority of the worms treated with the same concentration of caffeine after the L2 stage eventually developed into the adult stage, although their growth rate was significantly slower than the non-caffeine-treated controls and some worms also arrested as larvae (Fig. 3). These results indicate that although L1-stage larvae were the most susceptible to caffeine treatment, later-stage larvae were also partially susceptible.
To clarify that the larval developmental arrest after caffeine treatment was not caused by starvation due to blocking of pumping, we measured the pharyngeal pumping rate. Both L1-stage worms and L4-stage worms cultured in the presence of 30 mM caffeine for 24 h still maintained pumping although the rate was reduced to 69% and 64%, respectively, of noncaffeine-treated controls (Supplementary Fig. S1). These results clearly indicate that the larval arrest after 30 mM caffeine treatment was not caused by starvation due to blocking of pumping although this reduction of pumping rate might have affected the slow growth rate of worms treated with caffeine since the L2, L3, and L4-stages (Fig. 3).
To study the effects of caffeine treatment on global gene expression changes in
Synchronized L1-stage larvae were treated with RNAi of
We also examined RNAi of
In humans, caffeine is mainly metabolized by CYP1A2 in the liver (Kot and Daniel, 2008), while neonates have a significantly reduced capacity for caffeine metabolism (Brent et al., 2011). This raises the question of whether insufficient expression of
In this study, the effects of caffeine on developmental process and the expression of
We consider that early larval developmental arrest observed after 30 mM of caffeine treatment was not caused by starvation due to blocking of pumping because pumping was slowed but not completely blocked in this condition, although slow pumping rate might have affected growth rate of worms.
Two caffeine-resistant mutants were reported previously:
In summary, caffeine can interfere with
. Effects of caffeine on
Caffeine treatment | Brood size | Embryonic lethality (%) | Percent larval development (%) |
---|---|---|---|
0 mM | 250 ± 28.1 (100%) | 2.54 ± 3.00 | 95.2 ± 5.63 |
10 mM | 185 ± 22.2 (74.0%) | 1.64 ± 9,83 | 76.9 ± 15.1 |
20 mM | 167 ± 57.5 (66.8%) | 27.2 ± 11.2 | 66.0 ± 11.3 |
30 mM | 79.1 ± 27.3 (31.6%) | 50.2 ± 13.7 | 22.7 ± 5.78 |
10 synchronized L4-stage hermaphrodites were treated with different concentrations of caffeine after which brood size, F1 embryonic lethality, and F1 percent larval development were measured, as described in “Materials and Methods”.
. List of
Coding sequence | Gene name | Chromosome | Fold increased | |
---|---|---|---|---|
C49G7.8 | 5 | 33.2 | 0.0113 | |
K09D9.2 | 5 | 32.5 | 0.0211 | |
K07C6.5 | 5 | 28.9 | 0.0071 | |
C03G6.15 | 5 | 28.5 | 0.0084 | |
B0213.15 | 5 | 10.0 | 0.0056 | |
F08F3.7 | 5 | 8.3 | 0.0054 | |
R04D3.1 | X | 7.9 | 0.0181 | |
K07C6.3 | 5 | 6.5 | 0.0233 | |
C50H11.15 | 5 | 3.4 | 0.0027 | |
C26F1.2 | 5 | 3.3 | 0.0125 | |
F42A9.5 | 4 | 2.9 | 0.0146 | |
F41B5.2 | 5 | 2.8 | 0.0517 | |
F14F7.3 | 3 | 2.7 | 0.0348 | |
K07C6.4 | 3 | 2.4 | 0.0062 | |
K09A11.2 | X | 2.4 | 0.0383 | |
F44C8.1 | 5 | 2.4 | 0.04 | |
B0213.12 | 5 | 2.4 | 0.051 | |
C49C8.4 | 4 | 2.2 | 0.0246 | |
F41B5.7 | 5 | 2.2 | 0.0495 | |
F42A9.4 | 4 | 2.1 | 0.0161 | |
K09A11.3 | X | 2.0 | 0.0185 | |
F41B5.4 | 5 | 2.0 | 0.0337 | |
T10B9.4 | 2 | 2.0 | 0.0128 | |
E03E2.1 | X | 2.0 | 0.0147 |
*Only
. Relative expression levels of
Caffeine (mM) | Treated stage | ||||
---|---|---|---|---|---|
0 | L1 | 1.0 | 1.0 | 1.0 | 1.0 |
30 | L1 | 217 ± 0.6* | 2168.0 ± 18.8* | 2180.9 ± 4.9* | 4042.2 ± 45.4* |
0 | L4 | 1.0 | 1.0 | 1.0 | 1.0 |
30 | L4 | 8.4 ± 0.3* | 168.8 ± 4.2* | 1029.5 ± 3.7* | 97.9 ± 1.7* |
Real time RT-PCR was performed with total RNA extracted from synchronized L1-stage or L4-stage worms grown with 30 mM caffeine or without caffeine for 24 h as described in “Materials and Methods”.
Mol. Cells 2015; 38(3): 236-242
Published online March 31, 2015 https://doi.org/10.14348/molcells.2015.2282
Copyright © The Korean Society for Molecular and Cellular Biology.
Hyemin Min1,3, Ichiro Kawasaki1,3, Joomi Gong1, and Yhong-Hee Shim1,2,*
1Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Korea, 2Institute of KU Biotechnology, Konkuk University, Seoul 143-701, Korea, 3These authors contributed equally to this work.
Correspondence to:*Correspondence: yshim@konkuk.ac.kr
Intake of caffeine during pregnancy can cause retardation of fetal development. Although the significant influence of caffeine on animal development is widely recognized, much remains unknown about its mode of action because of its pleiotropic effects on living organisms. In the present study, by using
Keywords: caffeine,
1,3,7-trimethylxanthine, commonly known as caffeine, is one of the most popular drugs in the world. It comes from coffee beans, tea leaves, kola nuts, cacao pods, and so on (Gilbert et al., 1976). People ingest caffeine in one form or another every day, and it has become very popular in our daily lives. Recently, caffeine consumption has been increasing even among teenagers. Caffeine is known to enhance physiological functions in humans including sharpening our mind, improving athletic performance, and many other health improvements (Reviewed in Weinberg and Bealer, 2002). However, caffeine is an addictive drug. Although its effects are milder than those of other addictive drugs such as amphetamines, cocaine, and heroin, overlapping channels mediate the signal (Nehlig, 1999), which means that caffeine can also cause medical concerns.
Research revealed that caffeine consumption both before and during pregnancy increased the risk of spontaneous abortion (Cnattingius et al., 2000) and births classified as ‘small for gestational age’ (Hoyt et al., 2014). Maternal caffeine intake of over 300 mg/day doubled the risk of miscarriage compared with that of 151 mg/day (Giannelli et al., 2003). Such results suggest that intake of high doses of caffeine during pregnancy is a risk factor for fetal growth retardation. Therefore, understanding the basis of developmental defects caused by caffeine intake is an emerging issue, considering the fact that caffeine is now intertwined in the daily life of most people. Caffeine is metabolized in the liver by cytochrome P450 (CYP450) and excreted as urine in mammals (Kot and Daniel, 2008). The half-life of caffeine ranges from 3 to 7 h in adult plasma. Interestingly, the half-life is prolonged during pregnancy, in which it can be more than 10 h (Knutti et al., 1981). In addition, neonates have a greatly reduced capacity to metabolize caffeine, and it is excreted into the urine in largely unmetabolized form until hepatic metabolism becomes activated (Brent et al., 2011). Mammals contain 51 CYP family members, which are grouped into 10 subfamilies (Nelson et al., 2004). Among them, CYP1 to CYP4 are the drug-metabolizing families, and caffeine has been shown to be metabolized by CYP1A2 in mammals (Kot and Daniel, 2008). CYP450s render drugs more water-soluble so they can be excreted more easily in the urine or bile, and thereby, the drugs are processed by oxidative metabolism. Thus, CYP450s are able to promptly reduce the effects of drugs. The reduced capacity of neonates to metabolize caffeine therefore suggests that insufficient neonatal
Methods for the maintenance and handling of
L4-stage N2 hermaphrodites were individually cloned onto either caffeine-containing or control NGM agar plates and grown at 20°C. They were transferred to new plates in 24 h intervals for three days to allow embryo production. Laid embryos were considered dead if they did not hatch after 48 h at 20°C. Brood size was calculated as the total number of non-hatched and hatched embryos produced by a single mother hermaphrodite. Embryonic lethality was calculated as the percentage of non-hatched embryos among the total number of embryos produced. Percent larval development was calculated as the percentage of larvae that reached the adult stage among the total number of hatched embryos, as previously described (Kawasaki et al., 2013). The broods of 10 mother hermaphrodites were examined to calculate the above values at each concentration of caffeine treatment.
Synchronized L1-stage worms were incubated on the plate containing 0 or 30 mM caffeine in the absence of food,
Synchronized L1-stage worms were grown on NGM agar plates with or without caffeine for 3 days. Total RNA was extracted from the worms with Trizol reagent (Sigma, USA) and purified, after which the first strand cDNA was synthesized by M-MLV reverse transcriptase (Gibco BRL, USA) using oligo-dT primer (Promega, USA).
Synchronized L1-stage or L4-stage worms were grown on NGM agar plates with or without caffeine for 24 h. Total RNA was extracted from the worms with Trizol reagent (Sigma, USA), purified, and reverse transcribed with M-MLV reverse transcriptase (Gibco BRL, USA) using oligo-dT primer (Promega, USA) to synthesize the first strand cDNA. Respective cDNA products were PCR-amplified using the following primers:
RNAi analysis was performed using the “RNAi-by-soaking” method, as described previously (Maeda et al., 2001). N2 worms were synchronized at the L1 larval stage in the absence of food, after which they were soaked for 24 h in each of the double-strand RNA solutions transcribed
All experiments were repeated more than three times for statistical evaluation of data.
As a preliminary study, we examined growth rates of worms after treatment of 0, 5, 10, 15, 20, and 30 mM caffeine. We found that effects of caffeine were dosage-dependent, and determined three different concentrations of caffeine, 10, 20, and 30 mM as mild, moderate, and critical doses, respectively. To examine the effects of caffeine on the developmental process of
The effect of caffeine on the larval development of
To confirm that the larvae arrested with the 30 mM caffeine treatment indeed did not reach an adult stage, the expression of
Next, the effect of caffeine during different stages of larval development was examined by treating the worms with 30 mM caffeine starting from the L1, L2, L3, L4, or young adult stage (Fig. 3). Worms treated with 30 mM caffeine since the L1 stage were found to be mostly arrested at the L1 larval stage, as observed above. In contrast, the majority of the worms treated with the same concentration of caffeine after the L2 stage eventually developed into the adult stage, although their growth rate was significantly slower than the non-caffeine-treated controls and some worms also arrested as larvae (Fig. 3). These results indicate that although L1-stage larvae were the most susceptible to caffeine treatment, later-stage larvae were also partially susceptible.
To clarify that the larval developmental arrest after caffeine treatment was not caused by starvation due to blocking of pumping, we measured the pharyngeal pumping rate. Both L1-stage worms and L4-stage worms cultured in the presence of 30 mM caffeine for 24 h still maintained pumping although the rate was reduced to 69% and 64%, respectively, of noncaffeine-treated controls (Supplementary Fig. S1). These results clearly indicate that the larval arrest after 30 mM caffeine treatment was not caused by starvation due to blocking of pumping although this reduction of pumping rate might have affected the slow growth rate of worms treated with caffeine since the L2, L3, and L4-stages (Fig. 3).
To study the effects of caffeine treatment on global gene expression changes in
Synchronized L1-stage larvae were treated with RNAi of
We also examined RNAi of
In humans, caffeine is mainly metabolized by CYP1A2 in the liver (Kot and Daniel, 2008), while neonates have a significantly reduced capacity for caffeine metabolism (Brent et al., 2011). This raises the question of whether insufficient expression of
In this study, the effects of caffeine on developmental process and the expression of
We consider that early larval developmental arrest observed after 30 mM of caffeine treatment was not caused by starvation due to blocking of pumping because pumping was slowed but not completely blocked in this condition, although slow pumping rate might have affected growth rate of worms.
Two caffeine-resistant mutants were reported previously:
In summary, caffeine can interfere with
. Effects of caffeine on
Caffeine treatment | Brood size | Embryonic lethality (%) | Percent larval development (%) |
---|---|---|---|
0 mM | 250 ± 28.1 (100%) | 2.54 ± 3.00 | 95.2 ± 5.63 |
10 mM | 185 ± 22.2 (74.0%) | 1.64 ± 9,83 | 76.9 ± 15.1 |
20 mM | 167 ± 57.5 (66.8%) | 27.2 ± 11.2 | 66.0 ± 11.3 |
30 mM | 79.1 ± 27.3 (31.6%) | 50.2 ± 13.7 | 22.7 ± 5.78 |
10 synchronized L4-stage hermaphrodites were treated with different concentrations of caffeine after which brood size, F1 embryonic lethality, and F1 percent larval development were measured, as described in “Materials and Methods”..
. List of
Coding sequence | Gene name | Chromosome | Fold increased | |
---|---|---|---|---|
C49G7.8 | 5 | 33.2 | 0.0113 | |
K09D9.2 | 5 | 32.5 | 0.0211 | |
K07C6.5 | 5 | 28.9 | 0.0071 | |
C03G6.15 | 5 | 28.5 | 0.0084 | |
B0213.15 | 5 | 10.0 | 0.0056 | |
F08F3.7 | 5 | 8.3 | 0.0054 | |
R04D3.1 | X | 7.9 | 0.0181 | |
K07C6.3 | 5 | 6.5 | 0.0233 | |
C50H11.15 | 5 | 3.4 | 0.0027 | |
C26F1.2 | 5 | 3.3 | 0.0125 | |
F42A9.5 | 4 | 2.9 | 0.0146 | |
F41B5.2 | 5 | 2.8 | 0.0517 | |
F14F7.3 | 3 | 2.7 | 0.0348 | |
K07C6.4 | 3 | 2.4 | 0.0062 | |
K09A11.2 | X | 2.4 | 0.0383 | |
F44C8.1 | 5 | 2.4 | 0.04 | |
B0213.12 | 5 | 2.4 | 0.051 | |
C49C8.4 | 4 | 2.2 | 0.0246 | |
F41B5.7 | 5 | 2.2 | 0.0495 | |
F42A9.4 | 4 | 2.1 | 0.0161 | |
K09A11.3 | X | 2.0 | 0.0185 | |
F41B5.4 | 5 | 2.0 | 0.0337 | |
T10B9.4 | 2 | 2.0 | 0.0128 | |
E03E2.1 | X | 2.0 | 0.0147 |
*Only
. Relative expression levels of
Caffeine (mM) | Treated stage | ||||
---|---|---|---|---|---|
0 | L1 | 1.0 | 1.0 | 1.0 | 1.0 |
30 | L1 | 217 ± 0.6* | 2168.0 ± 18.8* | 2180.9 ± 4.9* | 4042.2 ± 45.4* |
0 | L4 | 1.0 | 1.0 | 1.0 | 1.0 |
30 | L4 | 8.4 ± 0.3* | 168.8 ± 4.2* | 1029.5 ± 3.7* | 97.9 ± 1.7* |
Real time RT-PCR was performed with total RNA extracted from synchronized L1-stage or L4-stage worms grown with 30 mM caffeine or without caffeine for 24 h as described in “Materials and Methods”..
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