Effect of dietary Ca and Cd level of pregnant rats on reproduction and on dam and progeny tissue mineral concentrations.

Pregnant Sprague-Dawley rats were used to determine the effects of the addition of 200 ppm of Cd (as CdCl2) to the diet factorially with two levels of dietary Ca (0.07% and 0.96%) on reproductive performance, concentrations of Cd, Cu, Fe, Zn, Ca and Mg in dam liver and kidney and in newborn progeny. High Cd significantly increased liver and kidney Cd, Zn and Ca and decreased liver Fe. High dietary Ca partially protected against accumulation of Cd in liver and kidney but had no effect on concentration of other elements. Number of live or stillborn pups per litter was not significantly affected by diet but high Cd significantly reduced pup birth weight. No grossly abnormal pups were noted. Concentration of Cd in bodies of newborn pups was increased approximately 8.6-fold by high Cd in the diet of dams fed the 0.07% Ca-diet and 3.8-fold by high-Cd in the diet of dams fed the 0.96% Ca diet. Pup, Zn, Cu and Fe contents were significantly decreased and Ca was significantly increased by high-Cd in the maternal diet whereas pup Mg content was unchanged. Maternal Ca intake had no effect on concentration of Zn, Cu, Fe or Ca in newborn pups. The biological importance of the alteration in maternal and fetal tissue concentration of Zn, Cu and Fe by high-Cd maternal diets is unknown.     

Regulation of the ontogeny of rat liver metallothionein mRNA by zinc

To investigate the role of metals in the regulation of the ontogenic expression of rat liver metallothionein (MT) mRNA, the concentrations of zinc, MT and MT mRNA were determined in livers of fetal and newborn rats from dams which were fed with a control or zinc-deficient or copper-deficient or iron-deficient diet from day 12 of gestation. The liver samples were analyzed for MT-mRNA levels using a mouse MT-I cRNA probe. Although the newborn hepatic levels of each metal (zinc or copper or iron) was specifically reduced corresponding to the respective mineral deficiencies, the hepatic concentrations of total MT and MT-I mRNA were significantly decreased only in pups born from zinc-deficient dams. Injection of the zinc-deficient newborn pups with 20 mg Zn as ZnSO4/kg restored with MT-I mRNA levels to slightly above control values within 5 h of injection. The hepatic zinc, MT and MT-I mRNA levels were observed to increase significantly in control fetal rat liver on days 17-21 of gestation but there were little changes in either zinc or MT in fetal livers from zinc-deficient dams during the late gestational period. The MT-I mRNA level also did not show an increase on days 18 and 20 of gestation in zinc-deficient fetal liver as compared to controls. These results demonstrate a direct role of zinc in hepatic MT gene expression in rat liver during late gestation. Immunohistochemical localization of MT using a specific antibody to rat liver MT showed that the staining for MT in zinc-deficient pup liver was mainly in the cytosol in contrast to the significant nuclear MT staining observed in control newborn rat liver. The results suggest that maternal zinc deficiency has a marked effect not only in decreasing the levels of hepatic MT and MT-I mRNA but also in the localization of MT in newborn rat liver.     

Inducibility and amounts of metallothionein as influenced by cadmium and zinc in the developing rat

Information on the accumulation and/or depletion of Zn in metallothionein (MT) of rat fetus, rat pup, and maternal rat liver at various ages was obtained with pregnant rats fed a basal casein diet or this diet plus either 100 ppm Zn or 50 ppm Cd. Rats fed each of the respective diets were sacrificed on 12, 16, and 20 d of gestation and 0, 7, 14, and 28 d post-partum. No Cd was detected in the placenta or fetal tissue and the Cd did not affect the accumulation of Zn in the fetal MT, but it did increase the Zn content in liver MT of the dams. Very little Zn in MT was found on day 12 of gestation, but Zn rapidly increased in MT to a maximum at time of birth. The accumulation of Zn in MT was independent of the diet for the fetuses, but the Zn accumulation in the dam and pup tissues was diet dependent. In order to study age-dependent difference in the inducibility of MT, newborn, 5-week-old, or 24-week-old rats were injected with zinc at the levels of 0, 3, 6, or 9 mg/kg and 5 h later injected with³⁵S-cystine. In rats sacrificed 1 h later, the amount of radioactivity in liver MT demonstrated that this protein in older animals was more readily induced by Zn than in younger animals.     

Effect of dietary caffeine and zinc on the activity of antioxidant enzymes,zinc, and copper concentration of the heart and liver in fast-growing rats

The purpose of this study was to determine the relationship between concentrations of Zn and Cu and the activities of superoxide dismutase and glutathione peroxidase in the heart and liver of young rat pups whose dams were fed a diet supplemented with caffeine and/or Zn. Four groups of dams with their newborn pups were fed one of the following diets for 22 d: 20% protein basal diet; the basal diet supplemented with caffeine (2 mg/100 body wt); the basal diet supplemented with Zn (300 mg/kg diet); or the basal diet supplemented with caffeine plus Zn. The Cu levels in the livers of the pups were decreased by maternal intake of the caffeine and Zn diet. The maternal intake of the caffeine diet increased Mn-superoxide dismutase (MnSOD) activity and Cu, Zn-superoxide dismutase (CUZnSOD) in the heart of the pups. On the other hand, the activity of Cu,ZnSOD was significantly reduced in the liver of pups whose dams consumed a caffeine, Zn, or caffeine plus Zn diet. Cu, ZnSOD activity in the liver of the pups seems to be correlated with Cu levels in the tissue. Selenium-dependent glutathione peroxidase (GSH-Px) activities in the heart and liver showed no difference among the groups. The effect of dietary caffeine and/or Zn on the activity of antioxidant enzymes in the heart and liver were different in young rats. The activities of these enzymes in the heart were lower than in the liver of 22-d-old rats. Our experiments indicate that the heart has limited defenses against the toxic effects of peroxides when compared to the liver.     

(Copper, zinc)--thionein in pig liver.

The concentrations of zinc and/or copper in the hepatic metallothionein of young (1–11 days, and 8 week-old) pigs correlate with the concentration of whole liver zinc. In the livers of adult animals only thionein-bound zinc correlates with total zinc. Although thionein-bound copper correlates with total copper in the livers of developing pigs, it shows a higher degree of correlation with whole liver zinc.Hepatic (copper, zinc) -thioneins, from either the young or the adult pig, in which the Cu : Zn ratio is high, are denatured on columns of DE cellulose, or during preparative electrophoresis. Preparations in which the Cu : Zn ratio is lower are resolved by ion-exchange chromatography into three forms, all of which contain both cations, but in differing ratios. Electrophoresis of a (copper, zinc) thionein that contains the two cations in 1 : atomic ratio yields zinc-thionein as a single distinct molecular species.     

Influence of cadmium on the distribution of the essential trace elements zinc and copper in the liver and kidneys of rats

Cd induced changes of Zn and Cd distribution in the liver and kidneys were studied in relation to Cd metallothionein (MT) synthesis. Wistar male rats were given CdCl₂ by sc injection of .8, 1.5, and 3.0 mg Cd/kg three times a week for three weeks. Cd levels of liver and kidneys increased with the increment of Cd dosage and 80–90% of Cd was found in the cytosol. The MT fractions contained 80–89% cytosolic Cd in the liver and 55–75% Cd in the kidneys. Zn concentrations in the liver increased following Cd administration, But Zn in the kidneys showed only slight increase. There was a distinct decrease of Cu concentration in the liver of the 3.0 mg group. In contrast, Cu concentrations in the kidneys increased about three times in the .8 and 1.5 mg Cd groups, but Cu in the 3.0 mg group showed only 1.5 times increase. The changes of these metal concentrations were observed mainly in the cytosol. Non-MT-Cd in the kidneys was maximum in the 1.5 mg group, but the 3.0 mg group showed significant decrease. In parallel with this decrease of Cd, Cu and Zn in the kidneys showed similar decrease. When the kidneys are injured, Zn and Cu appear to leak from this organ.     

On metallothionein,cadmium, copper and zinc relationships in the liver and kidney of adult rats

1. A short-term exposure of adult Wistar rats to Cu (50 μg/ml) and Cd (10.0 μg/ml drinking water) caused significant changes in the subcellular concentrations of Cd, Cu, Zn and metallothionein (MT) in the liver and kidney; the concentrations were close to the physiological values, however.2. To establish a relationship between these changes in the subcellular concentrations of Cd, Cu, Zn and the level of MT in the post-mitochondrial fraction of the liver and kidney, the analytical data (N = 42) were subjected to the multiple regression analysis.3. The analysis showed that MT synthesis in the liver was principally induced by small amounts of Cd (0.32–1.4 μg/g wet wt) whereas in the kidney a level of MT in the post-mitochondrial fraction correlated positively with the renal Cd and Cu, as well as with the level of this protein in the liver.4. The above results together with the positive correlation between the level of MT in the post-mitochondrial fraction and the concentration of Cu in this fraction, as well as the fact that under normal physiological conditions the capacity of MT (β-domain) in the liver and kidney was sufficient to bind 50–100% of the total post-mitochondrial Cu suggest that MT, first induced by small amounts of Cd, may be involved in the metabolism of Cu.     

Discriminative uptake of metals by the liver and its relation to induction of metallothionein by cadmium, copper and zinc

1. Disappearance from plasma and uptake by the liver of cadmium (Cd), copper (Cu) and zinc (Zn) were examined with a view to studying the biological discrimination between essential and non-essential heavy metals. 2. Cd injected intravenously at a single dose of 0.8 mg/kg body wt disappeared from rat plasma rapidly within about 10 min, while Cu and Zn injected at the same dose disappeared slowly in plasma and decreased to the control level after about 3 hr. 3. Uptake of Cd by the liver corresponded well with the rapid disappearance from plasma, while Cu and Zn accumulated slowly in the liver and their concentrations started to increase after their plasma concentrations had decreased. 4. Metallothionein was induced in the liver at a similar time course for the three metals, suggesting the presence of discriminative uptake processes by the liver with similar or the same detoxification mechanisms through induction of metallothionein.     

Influence of maternal mineral deficiency on the hepatic metallothionein and zinc in newborn rats

The effects of maternal Zn, Cu, or Fe deficiencies during late gestation on hepatic levels of metals and metallothionein (MT) and the binding of Zn and Cu to protein fractions were investigated in newborn rats. Timed pregnant rats were fed one of the following diets: Zn deficient (Zn-D), Cu-D, Fe-D, or control from day 12 of gestation until birth. The specific nutritional deficiency status of the dams was confirmed by low plasma levels of the deficient metal. Livers from pups were analyzed for MT, metal content, and metal-protein binding. Maternal Zn-D resulted in a greater than 50% reduction of hepatic MT levels in pups, whereas Cu-D and Fe-D had no significant effects. Pups in each deficient group showed a significant decrease in the hepatic levels of the respective metals. Fractionation of hepatic cytosols from the pups by Sephadex G-75 gel filtration showed that in both Fe-D and Cu-D pups the respective metals were depleted from the high molecular weight protein fraction, whereas in Zn-D pups the Zn was depleted mainly from the MT fraction (Ve/V0 approximately 2). Incorporation of [35S] cysteine into MT fractions was significantly lower in Zn-D pups as compared with control pups. These results indicate that there is a specific effect of the maternal Zn-D on the hepatic storage of Zn as MT in newborn rats.     

Concentrations of cadmium,zinc, copper,iron, and metallothionein in liver and kidney of nonhuman primates

To evaluate the species specificity of Cd accumulation and the relationship of Cd with other essential metals and metallothionein (MT), the concentrations of Cd, Zn, Cu, and Fe in the liver and kidney and the MT concentrations in the soluble fractions of the liver and kidney were determined in Cd-uncontaminated nonhuman primates (11 species, 26 individuals) kept in a zoo and two wild-caught Japanese macaques. The compositions of metal-binding proteins in the soluble fractions were also investigated by high-performance liquid chromatography (HPLC). The hepatic Cd concentration was 0.03–14.0 μg/g and the renal Cd concentration was 0.35–99.0 μg/g, both varying greatly and being higher in nonhuman primates, which were more closely related to man. The hepatic Zn concentration was 24.0–176 μg/g and the renal Zn concentration was 13.5–138 μg/g, showing 7- to 10-fold differences, and a correlation (r=0.558, p<0.01) was found between renal Zn and renal Cd concentrations. It was proved that in the liver, MT is more closely correlated with Zn (r=0.795, p<0.001) than with Cd (r=0.492, p<0.01) and that in the kidney MT is correlated with both Cd (r=0.784, p<0.001) and Zn (r=0.742, p<0.001). HPLC analysis of metals bound to MT-like protein in chimpanzees, de Brazza’s monkeys, and Bolivian squirrel monkeys showed that more than 90% of Cd in both the liver and kidney, approx 40% of Zn in liver and 28–69% of Zn in kidney were bound to MT-like protein. The higher percentage Zn was bound to high-molecular protein.     

Variation in the amounts of hepatic copper, zinc and metallothionein mRNA during development in the rat.

Amounts of hepatic metallothionein mRNA were assessed in RNA from foetal and neonatal rat livers by using dot-blot hybridization. Metallothionein mRNA began to increase about day 15 of gestation and reached a foetal maximum of 5-fold higher than adult values between 18 and 21 days of gestation. The amounts fell significantly for the first 3 days after parturition, and rose again to 6-fold above adult values 6 days after birth. By 15 days after birth the metallothionein mRNA had declined to adult amounts. In comparison, amounts of ornithine transcarbamoylase mRNA did not vary greatly during development. Hepatic zinc concentrations increased from day 14 of gestation to a maximum just before birth, and remained above adult values until 30 days after birth. From 14 days of gestation to 8 days after birth, hepatic copper concentrations were about 4-fold higher than in the adult, but a substantial increase (to about 9-fold higher than in the adult) occurs between 10 and 15 days after birth. CdCl2 administered to pregnant rats on day 18 of gestation was shown to block placental transfer of zinc, and we found decreased foetal hepatic zinc concentration after the CdCl2 treatment, but this failed to cause a significant decrease in metallothionein mRNA, suggesting that zinc may not be the primary inducer of hepatic metallothionein mRNA during foetal life.     

Interaction between copper and zinc in metal accumulation in rats with particular reference to the synthesis of induced-metallothionein

The effectiveness of Zn at moderating the pro-oxidant effects of Cu was evaluated in two rat models that differed in the route and mode of administration. The endpoints investigated included measurement of the concentrations of Cu, Zn, metallothionein and glutathione concentrations, as well as SOD and catalase activity, in liver, kidneys and intestine. In a sub-chronic animal model, the hepatic accumulation of Cu was achieved by administration of dietary Cu (1.8 g/kg solid diet) for 30 days after which oral Zn (6g/kg solid diet) was given. Cu treatment induced an increase in the hepatic and intestinal concentration of Cu of 66 and 455%, respectively, that was not associated with synthesis of metallothionein synthesis, but rather appeared to be related to the higher activity of SOD. Subsequent administration with Zn after dietary Cu induced an increase in the hepatic and intestinal metallothionein content of more twice and reduced the Cu content to control values. Thus, Zn could act as both a competitor for absorption on the luminal side of the intestinal epithelium inducing the synthesis of metallothionein. In the second animal model, we studied the effects of interaction between Cu and Zn administered by i.p. injection at the dose of 3 and 10mg/kg, respectively; Zn was administered subsequent to Cu overload. In this case, when Zn was administered, Cu was already deposited in tissues and thus there is no competition between two metals at the level of membrane transport. In this experimental model treatment with Cu alone induced liver metallothionein synthesis, and the subsequent treatment with Zn did not decrease the hepatic content of Cu. One explanation for these observations is that Zn induces the synthesis of metallothionein, which binds Cu for which it has a higher affinity. Moreover, after treatment with Zn, SOD activity in the liver decreases of almost 30% with respect to treatment with alone Cu, suggesting that Zn has a protective effect.     

Metabolism of copper and zinc in the liver and bone of the perinatal guinea-pig

Hepatic copper concentration in the guinea-pig increased markedly during the second-half of gestation, attaining a maximum shortly after birth; thereafter, concentration declined rapidly during the neonatal period. Changes in perinatal hepatic copper concentrations paralleled the binding of copper to a cytosolic metallothionein-like component, and the loss of hepatic copper in the neonates coincided with increases in serum copper concentrations. Zinc concentrations of the perinatal liver were low and showed no dramatic developmental changes. The humerus showed striking increases in zinc concentration with gestational age, attaining peak concentration before term and a marked depletion of tissue zinc during the neonatal period.     

Mercury,cadmium, zinc and copper concentrations and stable isotope ratios of carbon and nitrogen in tiger sharks (Galeocerdo cuvier) culled off Ishigaki Island,Japan

We analyzed Hg, Cd, Zn and Cu in the liver as well as Hg and stable isotope ratios of carbon and nitrogen (δ¹³C and δ¹⁵N) in muscle from tiger sharks (Galeocerdo cuvier) in Japan. The Hg concentration in the muscle increased slightly and proportionally with increases in body length, but the Hg concentration in the liver increased markedly after maturation (exceeding 2.7 m precaudal length). The Hg concentration in the liver of mature shark was higher than that in the muscle. The Cd concentration in the liver increased with increases in body length. On the other hand, the Zn and Cu concentrations in the liver decreased during the growth stage, but thereafter increased with increased Cd burden due to growth. The marked increase in hepatic Hg in mature sharks may be explained by the continuous intake of Hg via food, slower growth and Hg–Se complex formation. High concentrations of Zn and Cu in the liver of immature sharks and concomitant increases in Zn and Cu with the Cd burden in the liver of mature sharks may be explained by the physiological requirements of Zn and Cu during the growth stage, the induction of metallothionein synthesis due to the Cd burden and the subsequent binding of these metals to metallothionein. The δ¹⁵N and δ¹³C values decreased with increases in body length, suggesting a shift from coastal feeding to pelagic feeding with shark growth. The Hg and Cd concentrations tended to be negatively correlated with the δ¹⁵N and δ¹³C values as a result of the increase in Hg and Cd accumulation due to the growth and the decreases in δ¹⁵N and δ¹³C values due to the sift of feeding area.     

Gestational changes in concentrations of selenium and zinc in the porcine fetus and the effects of maternal intake of selenium

The effect of maternal dietary selenium (Se) and gestation on the concentrations of Se and zinc (Zn) in the porcine fetus were determined. Mature gilts were randomly assigned to treatments of either adequate (0.39 ppm Se) or low (0.05 ppm Se) dietary Se. Gilts were bred and fetuses were collected throughout gestation. Concentrations of Se in maternal whole blood and liver decreased during gestation in sows fed the low-Se diet compared to sows fed the Se-supplemented diet. Maternal intake of Se did not affect the concentration of Se in the whole fetus; however, the concentration of Se in fetal liver was decreased in fetuses of sows fed the low-Se diet. Although fetal liver Se decreased in both treatments as gestation progressed, the decrease was greater in liver of fetuses from sows fed the low-Se diet. Dietary Se did not affect concentrations of Zn in maternal whole blood or liver or in the whole fetus and fetal liver. The concentration of Se in fetal liver was lower but the concentration of Zn was greater than in maternal liver when sows were fed the adequate Se diet. These results indicate that maternal intake of Se affects fetal liver Se and newborn piglets have lower liver Se concentrations compared to their dams, regardless of the Se intake of sows during gestation. Thus, the piglet is more susceptible Se deficiency than the sow.     

Development of copper deficiency in neonatal mice

Dietary copper (Cu) deficiency was produced in Swiss albino mice to determine the temporal relationship between depletion of Cu and changes in the cardiovascular and nervous system. Dams were placed on a Cu-deficient diet 4 days after parturition. Half the dams were provided with deionized water and their offspring are referred to as Cu-deficient (-Cu). Half the dams were given cupric sulfate in their drinking water (20 microg Cu/mL) and their offspring are referred to as Cu-adequate (+Cu). At 6 weeks of age a sample of the -Cu mice were repleted with CuSO(4). Mice were sampled 1 day after birth and at weekly intervals for 7 weeks. Both +Cu and -Cu mice grew at the same rate: birth weight increased 16-fold at 6 weeks of age. Liver Cu more than doubled between 1 and 7 days of age. At 2 weeks of age -Cu mice were anemic (lower hematocrit and hemoglobin) and had lower liver Cu and plasma ceruloplasmin activity compared to +Cu mice. Liver Fe was not elevated in -Cu mice until 2 weeks after anemia developed. At weaning first signs of altered catecholamine metabolism included elevation of dopamine in both heart and spleen. Norepinephrine concentrations and content, in contrast, were not both lowered in -Cu mice until 5 weeks of age. Heart weight was first elevated in -Cu mice at 6 weeks of age and relative weight (mg/g body wt) at 4 weeks of age. Liver Cu concentration was lower in 1-week repleted mice than in +Cu mice. Anemia preceded the development of cardiac hypertrophy and altered catecholamine levels in -Cu mice.     

Associations of cadmium with zinc and copper in cancer cases and controls

This study was performed in order to search for possible associations between cadmium (Cd) and other metals~ Cd, zinc (Zn), and copper (Cu) levels of renal cortex and liver samples obtained from 196 cancer cases and 198 noncancer controls were analyzed in eight regional institutes in Japan. Cd, Zn, and Cu concentrations in liver and renal cortex of the selected cancer cases were compared to the controls whose localities, age, and sex were matched with the cancer cases. The cancer cases clearly showed higher accumulations of Zn in liver and renal cortex than the noncancer group. Cd showed a similar tendency, but there was no statistical significance between case and control subjects. There was no marked difference in Cu accumulation between the pairs. The best fit regression curve of Zn to Cd on a molar basis in renal cortex was obtained. Curvilinear regression equations of Zn to Cd for both cancer and noncancer groups were drawn. From these two equations, the Zn level of the cancer cases was found to reach a maximum when the Cd level showed 1.4 mmol/kg (157 pxg/g), whereas Zn in the noncancer group peaked at 1.6 mmol/kg (180 Ixg/g) of Cd in renal cortex. This may suggest that excessive Cd accumulation deprives binding sites of Zn in renal cortex when both metals are saturated.     

Postnatal changes in subcellular distribution of copper,zinc and metallothionein in the liver of bank vole (Clethrionomys glareolus): a possible involvement of metallothionein and copper in cell proliferation

1. Dramatic interdependent changes in the intracellular concentrations of copper (Cu), zinc (Zn) and metallothionein (MT) in the liver of bank voles during the first 30 days of their life were observed.2. The post-mitochondrial Cu, Zn and MT (ZnMT) abruptly decreased between 1 and 3 days following birth but the nuclear MT (CuMT) and Cu increased at the same time, suggesting that Cu displaced Zn already bound to MT in the cytoplasm and subsequently the complex CuMT was translocated to the nuclei.3. The nuclear Cu concentration reached the highest level (62–71% of the total tissue Cu) in the period from day 3 to day 20 post-partum, just prior to and during a rapidly growing liver.4. The data indicate that MT and Cu may be involved in the hepatocyte proliferation.     

Effect of zinc supplementation on metallothionein,copper, and zinc concentration in various tissues of copper-loaded rats

The present study was designed to investigate the effects of Zn administration on metallothionein concentrations in the liver, kidney, and intestine of copper-loaded rats. Male CD rats were fed a diet containing 12 mg Cu and 67 mg Zn/kg body wt. They were divided into either acute or chronic experimental protocols. Rats undergoing acute experiments received daily ip injections of either Cu (3 mg/kg body wt) or Zn (10 mg/kg body wt) for 3 d. Chronic experiments were carried out on rats receiving Cu ip injections on d 1, 2, 3, 10, 17, and 24, Cu injections plus a Zn-supplemented diet containing 5 g Zn/kg solid diet, or a Zn-supplemented diet alone. Rats injected Zn or Cu had increased MT concentrations in liver and kidney. Zn produced the most important effects and the liver was the most responsive organ. Rats fed a Zn-supplemented diet had significantly higher MT concentrations in liver and intestine with respect to controls. Increased MT synthesis in the liver may contribute to copper detoxification; the hypothesis of copper entrapment in enterocytes cannot be confirmed.     

Effects of pretreatment with cadmium on the discriminative uptake of subsequent cadmium, copper or zinc by the liver

1. Effects of pretreatment with cadmium (Cd) on the uptake by the liver of subsequent Cd, copper (Cu) and zinc (Zn) were examined at two different time intervals to elucidate the biological discrimination mechanism among metals of similar chemical properties. 2. Pretreatment with 0.3 mg Cd/kg body wt 6 hr but not 24 hr before a subsequent dose of 0.8 mg metal/kg body wt enhanced the disappearance rate from plasma and accumulation rate in the liver of Cu (and Zn) but not of Cd. 3. Synthesis of metallothionein was induced with different time-courses depending on the time interval between the pretreatment and subsequent treatment, which coincided with the accumulation curves for Cu (and Zn) but not for Cd. 4. Although uptake of Cd was not enhanced by any pretreatment, metallothionein synthesis was enhanced depending on the timing of pretreatment.