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Background: Anterior knee pain is very common in the young and active age groups but there are no significant studies on the normal population. Therefore, the objective of the study was to understand the relation between medial knee joint space to the development of knee pain and functional disability with special reference to the 20-45-year age group., Materials and Methods: 250 cases with mean age of 36.36 years presenting with knee pain for at least 1 month were included in this prospective study (July 2021 and June 2022) and were asked to self-complete questionnaires on knee pain which included KNEST, AKPQ, VAS and IPAQ. Patients underwent X-rays of bilateral knees in AP view with weight bearing and then medial and lateral knee joint spaces were calculated., Results: There was a higher incidence of knee pain in women compared to men. The average medial and lateral joint space widths (MJSW and LJSW) of the right knee were 4.22 mm and 4.57 mm, respectively. For the left knee it was 4.19 mm and 4.42 mm, respectively. There is a decrease in MJSW with increasing age, level of pain and BMI. Also, with an increase in physical activity, there was a higher incidence of knee pain., Conclusion: Overuse injuries are the main cause of knee pain in this age group. There is a significant association between medial joint space width and age, gender, level of pain, and BMI. Rising BMI patterns at a young age, leads to an increase in physical activity, which in turn leads to early knee pain and also predisposes to osteoarthritis., Competing Interests: Conflict of interestOn behalf of all the authors, the corresponding author states that there is no conflict of interest., (© Indian Orthopaedics Association 2023. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Background: The aim of the study was to uncover mortality risk utilizing a retrospective review at a level I trauma center in addition to demographic factors., Methods: Patients aged 65 and older with low-energy closed pelvic ring fractures treated non-operatively from 2007 to 2017 were queried from the level I trauma center database. Mortality rate and associated risks were calculated., Results: The average age of all the patients included in this study who sustained a low-energy pelvic fracture was 83.1 years (± 7.5; 66 - 97). The mean length of stay was 4.6 days (± 4.4; 0 - 37). The mean number of comorbidities was 2.2. The 1-year mortality rate was 23%. The relative risk (RR) of 1-year mortality for low-energy pelvic fractures for ages 65+ did not statistically differ compared to the US population in 2016 (6.6%) (RR: 1.0; 95% CI). The 2+ comorbidities showed a statistical significance in the pelvic fracture population with a P value of 0.037. Race, sex, discharge disposition and length of stay did not reach statistical significance (P > 0.05)., Conclusion: Low-energy pelvic injuries do not appear to increase rate of mortality compared to the US population. Fracture pattern, race, sex, discharge disposition and length of stay do not seem to have an effect on mortality. Elderly patients with an average age of 84.5 years and more than two comorbidities had higher rates of mortality; however, these patients were likely to sustain earlier mortality regardless of low-energy pelvic fracture., Competing Interests: None to declare., (Copyright 2019, Ghassibi et al.)

Neuromuscular-blocking agents are commonly used in laboratory animal research settings. Due to actions of cholinergic receptors at locations other than the motor end-plate, these agents have a strong propensity to modulate autonomic outflow and may therefore not be desirable in studies examining autonomic function. This study aimed to compare the effect of two non-depolarizing neuromuscular-blocking agents, pancuronium and cisatracurium, on blood pressure, heart rate and non-invasive indices of autonomic function (heart rate variability, systolic blood pressure variability and baroreflex sensitivity) under two different types of anaesthesia in Lewis rats. Pancuronium produced a profound vagolytic response characterized by tachycardia, reduction in heart rate variability and baroreflex sensitivity under urethane anaesthesia, and with minimal effect under isoflurane anaesthesia. Conversely, cisatracurium produced no evidence of vagolytic action under either urethane or isoflurane anaesthesia. Therefore, for studies interested in examining autonomic function, particularly baroreflex or vagal function, neuromuscular blockade would be best achieved using cisatracurium.

The current study reports the one-step synthesis and gelation properties of cyclohexane-based bis(acyl-semicarbazide) gelators with an additional -NH group incorporated into urea moieties and carrying hydrophobic chains of varying length (C8-C18). The gels exhibited thermoreversibility and could be tuned in the presence of anions at different concentrations in addition their the ultrasound-responsive nature, thus making them multi-stimuli-responsive. The combined experimental and computational study on these gels reveals that the balance between two noncovalent interactions, viz., hydrogen bonding between the amide groups in acyl-semicarbazide moieties and van der Waals forces between long hydrocarbon tails, is found to be the determining factor in the process of organogelation. A systematic increase in alkyl chain length leads to equilibrium between these two types of noncovalent forces that is manifested in the spectral and thermal properties of the gels. The H-bonding interactions dominated up to a certain chain length, and further increases in the alkyl chain length led to increased van der Waals interactions as observed by IR, XRD, and thermal studies. Computational calculations were carried out on dimer structures of C8-C18 to understand the variation in noncovalent forces responsible for aggregate formation in the gel state as a function of the alkyl chain length. The results indicate that both intermolecular and intramolecular hydrogen bonding stabilize the aggregate structures. Supramolecular aggregation in the gel state led to the viscoelastic nature of the gels, and the addition of anions led to the disruption of self-assembly, which was studied by rheology.

Altered autonomic control of the cardiovascular system in chronic kidney disease (CKD) contributes to an increased risk of cardiovascular events. The aim of the present study was to determine whether and when autonomic dysfunction occurs in a conscious, telemetered, rodent model of CKD. In Lewis polycystic kidney (LPK; n = 8) and Lewis (n = 8) rats, blood pressure (BP), heart rate (HR), HR variability (HRV), systolic BP variability (SBPV) and baroreflex sensitivity (BRS) were determined from 10 to 16 weeks of age. The LPK rats had higher systolic BP (average across all ages: 230 ± 10 vs 122.6 ± 0.3 mmHg; P < 0.001), increased SBPV (average across all ages: 13.9 ± 1.9 vs 5.2 ± 0.2 mmHg(2) ; P < 0.01) and reduced low-frequency HRV power (average across all ages: 1.5 ± 0.3 vs 2.6 ± 0.2 msec(2) ; P < 0.05). Between 10 and 12 weeks of age, SBPV increased twofold in the LPK rat (8.13 ± 1.05 vs 16.10 ± 1.31 mmHg(2) for 10 vs 12 weeks of age, respectively; P < 0.001), coinciding with an approximate 40% reduction in BRS (1.32 ± 0.14 vs 0.79 ± 0.11 ms/mmHg for 10 vs 12 weeks of age, respectively; P < 0.05). There was no difference in BRS between LPK and Lewis rats at 10 weeks of age; however, from 12 weeks onwards, BRS was reduced in LPK rats (0.75 ± 0.01 vs 1.17 ± 0.04 ms/mmHg; P < 0.01). Baroreceptor regulation of HR becomes impaired between 10 and 12 weeks of age in the LPK rat, coinciding with an increase in SBPV. Preventing baroreflex dysfunction in CKD may reduce SBPV and the associated mortality risks., (© 2013 Wiley Publishing Asia Pty Ltd.)

Background: The expression of P-glycoprotein (Pgp) is intimately associated with cancer development. In order to explore the therapeutic value of Pgp as a target for chemotherapy, we studied the effect of PSC 833 (PSC), a potent inhibitor of Pgp, on 1,2-dimethylhydrazine (1,2-DMH)-initiated colorectal carcinogenesis in rats., Materials and Methods: Male Fischer 344 rats, initiated with 1,2-DMH coupled with partial hepatectomy, were exposed to dietary 1% orotic acid for 22 weeks. They were then fed either the AIN93G basal diet (BD) or BD containing PSC (a daily dose of 15 mg/kg body weight) for 35 weeks., Results: PSC significantly inhibited colorectal tumor multiplicity by 53% and tumor burden by 74%. PSC-mediated inhibition was evident in tumors as small as 2 mm in diameter and remained effective throughout the course of tumor growth. Histological assessment showed that PSC significantly inhibited tumor progression to colorectal adenocarcinoma by 63%., Conclusion: Collectively, this study indicates that PSC inhibited experimental colorectal carcinogenesis initiated with 1,2-DMH in rats.

Studies in our laboratory on the role of P-glycoprotein (Pgp, coded by mdr1 gene) have led to the hypothesis that over-expression of Pgp is closely associated with the development of cancer. It was conceived therefore that inhibitors of Pgp should inhibit the development of cancer. We have reported that PSC833 (PSC), a potent inhibitor of Pgp, inhibits the development of liver cancer in rats. Similarly, based on the intrinsic over-expression of Pgp in experimental mammary carcinogenesis, we studied the effect of PSC on N-methyl-N-nitrosourea induced mammary cancer in female Sprague-Dawley rats. The study indicates that PSC at daily dietary doses of 15 (PSC15) and 30 mg/kg (PSC30) body wt resulted in dose-dependent inhibition of the incidence as well as the growth of mammary tumors. Compared with controls, PSC15 and PSC30 inhibited: (i) mean tumor multiplicity by 32 and 67%, (ii) median tumor burden by 46 and 93% and (iii) incidence of ulcerated tumors by 40 and 82%, respectively. Most remarkably, PSC delayed median tumor incidence by 8 weeks, and exerted a 100% inhibitory effect on the incidence of large tumors, 4 cm(3) and greater. In all the cases, although the inhibitory effect of PSC was evident at both doses, only PSC30 exhibited statistical significance. A possible compounding effect that was also observed in PSC30-treated rats was a decrease in body weight gain not attributed to diminished food consumption. All in all, consistent with recent reports, which have demonstrated inhibition of cancer development by compromising Pgp function, this study introduces a novel role for Pgp in breast cancer and potentially an unexplored therapeutic approach in treating the disease.

The present study explores the hypothesis that over-expression of P-glycoprotein (Pgp, product of mdr1) is intimately associated with liver cancer development and therefore inhibitors of Pgp should inhibit the development of liver cancer. Accordingly, we determined the effect of PSC833 (PSC), a potent inhibitor of Pgp, on experimental liver carcinogenesis in rats. To study the effects of PSC on liver cancer development, a daily dose of 30 mg PSC/kg body wt (PSC30) was chosen based on an initial dose-response experiment. Accordingly in experiment 1, PSC30 was fed to rats initiated by 1,2-dimethylhydrazine coupled with two-thirds partial hepatectomy and promoted for 22 weeks with 1% dietary orotic acid. Surprisingly, in contrast to our earlier observations in rats without hepatic nodules, in rats bearing hepatic nodules, PSC30 was found to be toxic. Because of this, PSC30 diet was discontinued after 5 weeks and the rats were transferred to basal diet (BD). The rats were killed 10 and 25 weeks thereafter. Cumulative results indicate that PSC30 exhibited a 40% decrease in the incidence of hepatocellular carcinoma (HCC; 15 of 18 in the BD group compared with eight of 17 in the PSC30 group; P = 0.08) coupled with significant reduction of tumor multiplicity (54%; P < 0.05) and tumor burden (61%; P < 0.005) compared with controls. In experiment 2, 15 mg PSC/kg body wt (PSC15) was fed for 20 weeks to rats similarly initiated and promoted for 35 weeks. PSC15 inhibited the incidence of HCC by 75% (four of four in the BD group compared to one of four in the PSC30 group; P = 0.15) and significantly reduced tumor burden by 55% (P < 0.05). The lack of statistical significance of inhibition on tumor incidence reflects the small sample size. Taken together the results indicate a possible intrinsic role for Pgp in liver cancer development and introduce another promising unexplored therapeutic approach in liver cancer treatment.

We report a serendipitous finding of urinary calculi in rats fed a semi-synthetic basal diet. This observation was made during ongoing studies to evaluate the inhibitory effect of PSC 833, a potent inhibitor of P-glycoprotein, on development of tumors in rodent tumor model systems. A large number of specific-pathogen-free (SPF) female Sprague-Dawley and SPF male Fischer 344 rats being fed the diet were euthanized when it became evident clinically that they were uremic. At necropsy, the renal pelvis, ureters, and urinary bladder contained numerous calculi. The presence of urinary calculi was determined to be related to the source of a Food Chemical Codex grade of choline bitartrate. Rats being fed the same basal diet containing the United States Pharmacopia grade of choline bitartrate failed to develop urinary calculi. Interestingly, rats treated with the P-glycoprotein inhibitor were at significantly reduced risk of developing urinary calculi. This finding highlights how something seemingly innocuous as a minor dietary constituent can have a profound impact and, thereby, affect experimental outcome.

Malignant astrocytomas have been found to express P-glycoprotein (Pgp, mdr1 gene product). It was hypothesized that in addition to conferring multidrug resistance, Pgp is intimately associated with the development of astrocytomas. Accordingly, we studied the effect of PSC 833 (PSC, Novartis), a potent inhibitor of Pgp, on the growth of Pgp-expressing astrocytoma cells. The results showed that in all the cell lines tested, PSC (10-60 microM) inhibited the growth as well as induced cell death. Cells exposed to PSC exhibited DNA ladder characteristic of apoptosis. PSC-induced cell death could be reversed by Z-VAD-fmk, a general caspase inhibitor, indicating that PSC-induced cell death was characteristic of caspase-mediated apoptosis. These results suggest a novel therapeutic strategy in the treatment of malignant astrocytomas by inhibitors of Pgp.

Previously, we have shown that Copenhagen (Cop) rats are highly resistant to the induction of putative preneoplastic, glutathione S-transferase 7-7- (GST 7-7) positive liver lesions following treatment with a modified resistant hepatocyte (RH) protocol. The objective of this study was to determine if resistance is inherited in a dominant or recessive manner and to derive an estimate of the number of genetic loci involved. We crossed male and female Cop rats with F344 rats to produce F1 offspring. Backcross rats were generated using female F1 rats and either Cop or F344 males, resulting in B1c and B1f generations, respectively. The male rats from all these crosses were initiated with diethylnitrosamine (200 mg/kg) at 7 to 8 weeks of age and were promoted 3 weeks later with the RH protocol (2-acetylaminofluorene and a two-thirds partial hepatectomy). The rats were sacrificed 3 weeks after the partial hepatectomy and their livers were sectioned and stained for GST 7-7-positive lesions. The susceptibility of F1 rats was in between Cop and F344 rats, having 21.7% +/- 2.0% (mean +/- SEM) of their liver volume occupied by lesions versus 4.2% +/- 0.8% for Cop and 53.0% +/- 5.8% for F344 rats. As expected, B1c rats had a volume of liver occupied by lesions that was in between the F1 and Cop rats at 13.5% +/- 1.6%. Surprisingly, B1f rats were similar to B1c rats in their resistance (9.1% +/- 2.1%). These results point to a complex, polygenic inheritance pattern that can be explained by a minimum of four loci, one of which shows recessive epistasis.

We recently have shown that selective growth of transplanted normal hepatocytes can be achieved in a setting of cell cycle block of endogenous parenchymal cells. Thus, massive proliferation of donor-derived normal hepatocytes was observed in the liver of rats previously given retrorsine (RS), a naturally occurring alkaloid that blocks proliferation of resident liver cells. In the present study, the fate of nodular hepatocytes transplanted into RS-treated or normal syngeneic recipients was followed. The dipeptidyl peptidase type IV-deficient (DPPIV(-)) rat model for hepatocyte transplantation was used to distinguish donor-derived cells from recipient cells. Hepatocyte nodules were chemically induced in Fischer 344, DPPIV(+) rats; livers were then perfused and larger (>5 mm) nodules were separated from surrounding tissue. Cells isolated from either tissue were then injected into normal or RS-treated DPPIV(-) recipients. One month after transplantation, grossly visible nodules (2--3 mm) were seen in RS-treated recipients transplanted with nodular cells. They grew rapidly, occupying 80--90% of the host liver at 2 months, and progressed to hepatocellular carcinoma within 4 months. By contrast, no liver nodules developed within 6 months when nodular hepatocytes were injected into the liver of recipients not exposed to RS, although small clusters of donor-derived cells were present in these animals. Taken together, these results directly point to a fundamental role played by the host environment in modulating the growth and the progression rate of altered cells during carcinogenesis. In particular, they indicate that conditions associated with growth constraint of the host tissue can drive tumor progression in vivo.

Background/aim: We have designed an experimental model in which transplantation of normal hepatocytes into rats previously treated with retrorsine (a naturally-occurring pyrrolizidine alkaloid) results in near-complete replacement of the recipient liver by donor-derived cells. Two/thirds partial hepatectomy was found to be essential for this process to occur. To probe this finding, in the present study we describe the kinetics of liver regeneration in response to partial hepatectomy in rats given retrorsine., Methods: Six-weeks-old male Fisher 344 rats received retrorsine (2 injections of 30 mg/kg each, i.p., 2 weeks apart), or the vehicle. Four weeks after the last injection, partial hepatectomy was performed and rats were killed at 1, 2, 3, 6, and 15 days thereafter., Results: At time zero, i.e. prior to partial hepatectomy, liver weight and total liver DNA content were significantly lower in retrorsine-treated animals compared to controls (DNA content: 19.2+/-1.7 vs. 25.7+/-1.1 mg/liver). Diffuse megalocytosis (enlarged hepatocytes) was present in the group exposed to retrorsine. By day 3 post-partial hepatectomy liver DNA content in control animals had more than doubled compared to day 1 values (20.2+/-1.5 vs. 8.8+/-1.2), while very little increase was seen in retrorsine-treated rats at the same time points (7.6+/-0.4 vs. 6.1+/-0.2). At 2 weeks after partial hepatectomy, total DNA content returned close to normal levels in the control group (26.9+/-1.0 mg/liver); however, the value was still very low in animals receiving retrorsine (9.1+/-0.7). Data on BrdU labeling were consistent with this pattern and indicated that DNA synthesis following partial hepatectomy was largely inhibited in the retrorsine group. Similarly, no mitotic response was observed in hepatocytes following partial hepatectomy in animals exposed to retrorsine., Conclusions: These results clearly indicate that retrorsine exerts a strong and persistent cell cycle block on hepatocyte proliferation. Further, these results are in agreement with the hypothesis that selective proliferation of transplanted hepatocytes in retrorsine-treated animals is dependent, at least in part, on the persistent cell cycle block imposed by the alkaloid on endogenous parenchymal cells.

Caloric restriction has been associated with a delay in the development of both spontaneous and induced neoplasia. In contrast, cycles of fasting/refeeding were shown by us and others to enhance the incidence of early lesions during chemical carcinogenesis in rat liver. The present, long-term study was undertaken to establish whether such a diffential effect would also extend to the later phases of cancer development, until the overt appearance of neoplasia. Male Fischer 344 rats were initiated with a single dose of diethylnitrosamine (DENA, 200 mg/kg i.p.) and starting 1 week later they were either exposed to three cycles of fasting (3 days) followed by refeeding (11 days) or were fed continuously. Seven weeks after DENA administration the rats were exposed to the resistant hepatocyte model of the liver tumor promotion protocol. All animals were killed 1 year after initiation. Incidence of hepatocellular carcinoma was 2-fold higher in the fasted/refed group compared with the controls (72 versus 36%). In addition, cancers were also larger and of higher histological grade in the former group, with one animal showing metastases to the lungs, while no metastases developed in control animals. Fasting caused a decrease in total liver DNA (from 25.2 +/- 1.1 to 16.5 +/- 1.1 mg after 3 days) which was associated with a decrease in hepatocyte labeling index and mitotic activity and high levels of single cell death (apoptosis). In contrast, a sharp increase in hepatocyte proliferation was observed on day 2 of refeeding and this was more pronounced in glutathione S-transferase 7-7 positive foci compared with surrounding liver (10.2 +/- 2.3 versus 4.6 +/- 0.8%). Such a proliferative wave was associated with a sharp decline in the incidence of cell death. It is concluded that fasting/refeeding performed early after initiation accelerates the development of chemically induced hepatocellular carcinoma in the rat.

The present study was designed to determine whether the resistant phenotype is acquired at the initiated cell stage itself or requires further exposure to a promoting regimen to express resistance. Male Fischer 344 rats were initiated with diethylnitrosamine (DENA) (200 mg/kg i.p.) and were subjected to either no further treatment or to the resistant hepatocyte (RH) model of liver tumor promotion. Six weeks later, the resistance of the focal lesions generated in these two groups to the mitoinhibitory effects of 2-acetylaminofluorene (2-AAF) was determined by subjecting the rats to two-thirds partial hepatectomy (PH) in the presence of a mitoinhibitory dose of 2-AAF (5 mg/kg i.p.) given at the time of PH. Labeling index was determined by administering multiple injections of [(3)H]thymidine. All rats were killed 48 h post-PH. While only a small percentage (23%) of the glutathione S-transferase-positive foci generated by DENA in the absence of an exogenous liver tumor promoting regimen were resistant to the mitoinhibitory effects of 2-AAF, a majority (85%) of the foci became resistant to 2-AAF following exposure to the RH model of liver tumor promotion. Further, initiated rats exposed to either 2-AAF or to CCl(4) alone, the two components of the RH model, resulted in 71% of the foci being resistant to the mitoinhibitory effects of 2-AAF. Similar patterns of results were obtained when the resistance of the foci to the mitoinhibitory effects of orotic acid, a liver tumor promoter and an inhibitor of DNA synthesis in normal hepatocytes, was monitored. These results suggest that the majority of initiated hepatocytes are not of resistant phenotype, however, they have acquired a unique ability to express resistance upon exposure to certain agents such as 2-AAF and CCl(4) or to a promoting regimen such as the RH model of liver tumor promotion.

Butyrate exerts potent anti-tumor effects by inhibiting cancer cell growth and inducing apoptosis. However, the molecular mechanisms mediating these effects remain largely unknown. Using the Caco-2 cell line, a well established model of colon cancer cells, our data show that butyrate induced apoptosis (maximum 79%) is mediated via activation of the caspase-cascade. A key event was the proteolytic activation of caspase-3, triggering degradation of poly-(ADP-ribose) polymerase (PARP). Inactivation of caspase-3 with the tetrapeptide zDEVD-FMK completely inhibited the apoptotic response to butyrate. In parallel, butyrate potently up-regulated the expression of the pro-apoptotic protein bak, without changing Caco-2 cell bcl-2 expression. Butyrate-induced Caco-2 cell apoptosis was completely blocked by the addition of cycloheximide, indicating the necessity of protein synthesis. However, when this inhibitor was added at a time point where bak expression was already enhanced (12 - 16 h after butyrate stimulation), it failed to protect Caco-2 cells against apoptosis. Taken together, these data provide evidence that the molecular events involved in butyrate induced colon cancer cell apoptosis include the caspase-cascade and the mitochondrial bcl-pathway.

Previously, we have shown that Copenhagen (Cop) rats are highly resistant to the induction of putative preneoplastic, glutathione S-transferase 7-7 (GST 7-7)-positive liver lesions following treatment with a modified resistant hepatocyte protocol. The objective of the current study was to establish the time course for the development of resistance and examine potential resistance mechanisms in Cop rats using F344 rats as susceptible controls. Male Cop and F344 rats (n = 25), 7-8 weeks of age, were initiated with diethylnitrosamine (200 mg/kg) and promoted 3 weeks later with four doses of 2-acetylaminofluorene (20 mg/kg) and a 2/3 partial hepatectomy (PH). Groups of rats from each strain were killed on days 2, 4, 7, 14 and 21 post-PH, 2 h after receiving bromodeoxyuridine. Cop livers contained similar numbers of GST 7-7-positive lesions to F344 livers on days 2 and 4 post-PH. The percent volume of liver occupied by these lesions did not differ between the strains on days 2, 4 and 7 post-PH. On day 14, however, approximately 29% of the liver volume in F344 rats was occupied by lesions, whereas in Cop rats this was significantly less (approximately 9%, P < 0.001). On day 21, lesions occupied approximately 58% of F344 rat livers and only approximately 6% of Cop livers. Despite these differences, the labeling index of hepatocytes was not significantly different between the strains at any time point, either within lesions or within surrounding normal liver. Furthermore, the apoptotic indices were not different between the strains at any time. However, differences were found in the extent of lesion remodeling (redifferentiation) and in the pattern of oval cell response following PH in Cop livers. By day 14 post-PH, approximately 76% of Cop liver lesions showed evidence of remodeling, compared with only approximately 14% of F344 lesions. The oval cell response to PH was equivalent in the two strains up to day 4 post-PH but by day 7, in F344 livers there was extensive migration of these cells into the liver parenchyma, whereas in Cop livers, the response remained localized to the portal regions. These results suggest that Cop resistance occurs at the promotion stage and not the initiation stage of carcinogenesis. Resistance appears not to be due to a lower proliferation rate nor to a higher apoptotic rate within Cop lesions. Precocious remodeling and/or a diminished oval cell response, however, may contribute to the resistance of Cop rats to the growth of GST 7-7-positive hepatic lesions.

Our previous studies indicated that glutathione S-transferase 7-7 (GST 7-7) positive foci induced after initiation have a lower threshold towards proliferative stimuli compared with surrounding hepatocytes. This observation would predict that persistent growth stimuli of low intensity could be very effective in promoting the emergence of focal lesions. To test this possibility, the present study was designed to determine the effect of 1/3 partial hepatectomy (PH) on the incidence and growth of foci in initiated rat liver. The rationale for using a 1/3 PH was that it is known to induce a proliferative response which is less intense but more prolonged compared with that elicited by 2/3 PH. Male Fischer 344 rats (110-120 g) were initiated with diethylnitrosamine (200 mg/kg, i.p.). Three weeks later 1/3 PH (median lobe), 2/3 PH (median and left lobes) or sham operation (SH) was performed. An additional group of initiated animals had the median lobe and the left lobe of the liver removed sequentially (1/3 + 1/3 PH), 3 weeks apart. All rats were killed 8 weeks after carcinogen administration. The results indicated that the number of GST 7-7 positive foci was similar in all groups; however, the percent area occupied by foci was increased in rats receiving 2/3 PH compared with SH (0.21 +/- 0. 08 versus 0.09 +/- 0.03). Interestingly, 1/3 PH was nearly as effective as 2/3 PH in stimulating the growth of foci (percent area 0.18 +/- 0.06 versus 0.21 +/- 0.08), although the magnitude of the stimulus is only half for the former group compared with the latter; peak labeling index was 19 +/- 6 with 1/3 PH compared with 40 +/- 2 with 2/3 PH. Moreover, the maximum increase in the size of foci (percent area 0.37 +/- 0.12) was achieved when the median and left lobes were removed sequentially, three weeks apart. These results indicate that persistent growth stimuli of low intensity can be very effective in promoting the growth of focal lesions.

Our earlier studies have shown that in normal hepatocytes, orotic acid (OA) inhibits DNA synthesis induced by several growth factors in vitro and after two-thirds partial hepatectomy (PH) in vivo. As in the normal liver OA induces an imbalance in nucleotide pools (specifically, an increase in uridine nucleotides, including deoxyuridine nucleotides, and a decrease in adenosine nucleotides, including ATP) and creation of this imbalance is crucial for the mitoinhibitory effects of OA, we hypothesized that ribonucleoside diphosphate reductase (RNR), a key enzyme in DNA synthesis that is regulated by nucleotide/deoxynucleotide levels, might be one of the targets for the inhibition of DNA synthesis by OA. To test this hypothesis, we subjected male Fischer 344 rats (130-150 g) to two-thirds PH in the absence or in the presence of OA (a 300-mg tablet of OA methyl ester implanted intraperitoneally at the time of two-thirds PH). The rats were killed at different times later, and their livers were processed for analysis of levels of RNR enzyme activity, protein, and mRNA transcripts. The results obtained indicated that treatment with OA resulted in a near-100% inhibition of RNR induced by two-thirds PH in rat liver, as monitored by enzyme activity and protein level. Furthermore, this inhibition was paralleled by a decrease in the mRNA transcripts for both the M1 and M2 subunits of RNR. Nuclear run-off assays indicated that this decrease in the levels of mRNA transcripts could not be attributed to an effect on transcription. However, administration of OA 20 h after two-thirds PH, when RNR mRNA transcripts were maximally induced, resulted in increased degradation of the RNR M1 and M2 subunits. Taken together, these results indicate that OA treatment decreases RNR levels induced by two-thirds PH, at the levels of enzyme activity, protein, and mRNA transcripts, and the decreased levels of mRNA transcripts appeared to be due to increased degradation of the transcripts.

Administration of either ammonia or glycine to both rats and mice results in an increased synthesis in the liver and urinary excretion of orotic acid. The two most relevant observations obtained are that carbamoyl phosphate synthesized inside the mitochondria is involved in the increased synthesis of orotic acid and that this latter process is almost completely abolished by cycloheximide and actinomycin D, inhibitors of protein and RNA synthesis. Orotic acid synthesis could be controlled by an induction-suppression mechanism. Inhibition of synthesis of excess orotic acid brought about by N-(phosphonacetyl)-L-aspartic acid but not by acivicin, suggests that glutamine-dependent cytosolic synthesis of carbamoyl phosphate, is not involved. Administration of ornithine together with glycine completely suppressed the synthesis of orotic acid, but promoted a twofold increase of urea excretion. The concentration of ornithine rather than that of carbamoyl phosphate or the activity of the enzymes involved, may represent a limiting factor controlling both the flux of ammonia in the urea cycle and the availability of mitochondrial carbamoyl phosphate for orotic acid synthesis. Two enzymes have been found to be induced by glycine: ornithine decarboxylase and aspartate transcarbamoylase (aspartate carbamoyltransferase). Both enzymes may contribute to the increase in orotic acid synthesis, aspartate transcarbamoylase more directly and ornithine decarboxylase by lowering the ornithine concentration. Ornithine decarboxylase activity was completely suppressed but that of aspartate transcarbamoylase was further increased by cycloheximide treatment. Inhibition of orotic acid biosynthesis by cycloheximide appears to be the result of a decreased availability in the cytosol of carbamoyl phosphate synthesized inside the mitochondria.

Copenhagen (Cop) rats are completely resistant to the chemical induction of mammary adenocarcinomas, but their susceptibility to hepatocarcinogenesis is virtually unknown. Rat liver is a well-characterized and easily manipulated tissue in which to study carcinogenesis. Therefore, if Cop rats are resistant to hepatocarcinogenesis, studies into resistance mechanisms may be feasible. Male Cop and F344 rats, 7-8 weeks old, were initiated using either N-nitrosodiethylamine (DEN) (200 mg/kg, i.p.) or a two-thirds partial hepatectomy (PH) followed by N-methyl-N-nitrosourea (MNU) (60 mg/kg, i.p.). The rats were then promoted using a modified resistant hepatocyte (RH) protocol (a combination of four doses of 2-acetylaminofluorene (2-AAF) and a single dose of CCl4 that provides a selective mitotic stimulus for initiated cells). Six weeks after initiation the rats were killed and liver sections were stained for glutathione S-transferase 7-7 (GST 7-7), a marker for putative preneoplastic hepatocytes. Cop rats were found to be highly resistant, having a approximately 9- and approximately 27-fold smaller percentage of liver area occupied by GST 7-7-positive foci than susceptible F344 rats following initiation by DEN and MNU respectively. Furthermore, gross liver nodules did not form in any of the Cop rats, whereas all F344 rat livers contained nodules. Hepatic necrosis caused by DEN during initiation, and CCl4 during promotion is necessary to stimulate compensatory hepatocyte division. We demonstrated that these agents do indeed increase serum transaminase levels and produce histologic evidence of necrosis in Cop rats. In order for liver foci to grow rapidly in the RH protocol, the surrounding normal hepatocytes must be mito-inhibited by 2-AAF. We found that the degree of mito-inhibition of normal hepatocytes by 2-AAF is the same in Cop and F344 rats. These results show that the Cop rat is highly resistant to the chemical induction of putative preneoplastic liver foci and nodules.

Background: Orotic acid (OA) is able to inhibit hepatocyte proliferation in vivo induced by 2/3 partial hepatectomy. The present studies were aimed at establishing: (i) whether OA also inhibits hepatocyte proliferation induced by a direct mitogen and, if so (ii) whether the stimulus provided by the mitogen is still expressed following transient inhibition by OA., Methods/results: In the first experiment male Wistar rats were injected with either lead nitrate (100 mumol/kg, i.v.) or saline and 20 h later some animals receiving the mitogen were also implanted with a 400-mg OA tablet (as OA-methyl ester. i.p.). Multiple injections of 3H-thymidine were given to each rat (50 microCi each, 6 h apart, i.p.) until 2 h before killing. All groups were killed 3 days after the initial treatment. Results indicated that OA almost completely inhibited hepatocyte DNA synthesis and labelling induced by lead nitrate (e.g. labelling index was 1.9 +/- 0.5% in the saline-treated group, 44.7 +/- 4.0% in the lead nitrate group and 1.4 +/- 0.3% in the group receiving lead nitrate + OA). Based on the above results, in a second experiment rats were given a similar dose of lead nitrate and a subset of animals was implanted 20 h later with a 400-mg OA tablet, as previously described. Multiple doses of 3H-thymidine were again given to each rat (20 microCi each, 6 h apart) until 2 h before killing. Animals from both groups were killed at 3, 6 or 8 days after lead nitrate. Results indicated that, while at day 3 lead nitrate-induced DNA synthesis was effectively inhibited by OA, at day 6 the proliferative response was resumed in the group receiving OA. Cumulative labelling index over 6 days was 30.3 +/- 1.4 in rats given the mitogen alone and 52.1 +/- 2.2 in the group exposed to lead nitrate + OA., Conclusions: These data indicate that: (i) OA is also able to inhibit hepatocyte proliferation induced by a direct mitogen such as lead nitrate; this, in turn, suggests that its inhibitory effect is not unique to the stimulus elicited by partial hepatectomy. (ii) The proliferative response triggered by the mitogen is not abolished by the transient (3-4 days) inhibitory phase imposed by OA. Possible mechanisms underlying these effects are considered in the discussion.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), is a rate-limiting enzyme in the biosynthesis of not only cholesterol but also a variety of non-sterol isoprenoids. It is subjected to multivalent feedback suppression by transcriptional and post-transcriptional control mechanisms mediated by sterols and non-sterol substances. In the present study, the effect of a plant isoprenoid, beta-carotene, on the expression of HMG-CoA reductase in rat liver was investigated. In control rats the hepatic levels of mRNA transcripts of HMG-CoA reductase increased following 2/3 partial hepatectomy with two peaks, one at 8 h and the other at 24 h. Administration of the carotenoid (70 mg/kg, given every alternate day for 3 consecutive weeks) partially inhibited the increase in the transcript level with a 50% reduction at 8 h and 30% reduction at 24 h post partial hepatectomy. Nuclear run-off assays with nuclei isolated from the resting liver and from livers of control rats and rats exposed to beta-carotene for 3 consecutive weeks and killed 8 h after partial hepatectomy indicated that beta-carotene did not inhibit the rate of transcription of HMG-CoA reductase gene. These observations suggest that beta-carotene regulates the expression of HMG-CoA reductase by some post-transcriptional mechanisms.

Caloric restriction causes a generalized decrease in growth rate and has been shown to delay the development of both spontaneous and induced neoplasia. In contrast to chronic food restriction, the extreme condition of fasting/refeeding is associated with an overall increase in cell turnover in several organs, including liver, compared with regular feeding. The present study was therefore designed to investigate the effect of complete food withdrawal followed by refeeding on the growth of hepatocyte nodules in initiated rat liver. Male Fischer 344 rats were given a single dose of diethylnitrosamine (DEN, 200 mg/kg i.p.) and then, starting 1 wk later, they were exposed to one or three cycles of fasting (3 days) followed by refeeding (11 days). The control group was fed continuously. Seven weeks after DEN administration all rats were subjected to the resistant hepatocyte model (2-acetylaminofluorene coupled with CCl4) and 2 weeks later 2/3 partial hepatectomy (PH) was performed. All animals were killed 2 weeks after surgery. At PH rats given one cycle of fasting/refeeding had significantly larger glutathione S-transferase 7-7-positive hepatic lesions compared with controls (mean area 0.73 +/- 0.04 versus 0.50 +/- 0.05 mm2, P < 0.025; mean percent area 25.6 +/- 3.2 versus 12.4 +/- 0.9, P < 0.005), while no significant change was observed in their number. The observed differences were more pronounced with three cycles of fasting/refeeding. A similar pattern of results was obtained at the time of killing. It is concluded that fasting/refeeding can exert a positive effect on the growth of rat hepatocyte foci and nodules, in contrast to the general inhibitory effect on carcinogenesis caused by food restriction.

Liver samples obtained at autopsy from patients with ornithine transcarbamylase (OTC) deficiency, a urea cycle disorder that is associated with high levels of orotic acid biosynthesis and excretion were analysed for nucleotide pools. As a control, liver samples from patients with a deficiency of mitochondrial carbamyl phosphate synthetase (CPS-I) which is not associated with increased levels of orotic acidurias were also analysed. The results show that liver tissue from OTC deficiency patients exhibited an increased ratio of uridine nucleotides to adenosine nucleotides, while in CPS-I deficiency patients, no such increase was noted. This study indicates that genetic disorders that are associated with increased loads of orotic acid exhibit abnormally high ratios of uridine to adenosine nucleotides in the liver. This type of imbalance is analogous to that seen in the liver of rats and mice exposed to an orotic acid supplemented or an arginine-deficient diet under liver tumor promoting conditions. It is likely that an imbalance in nucleotide pools may have a significant role in the pathophysiology associated with these disorders.

Chemoprevention of liver carcinogenesis by S-adenosyl-L-methionine (SAM) was studied in F344 male rats. The rats were given 1,2-dimethylhydrazine (1,2-DMH) 2 HCl (100 mg/kg, i.p.) 18 h after two-thirds hepatectomy. One week later they were fed a semisynthetic basal diet containing 1% orotic acid (OA) for 29 weeks. At this time the rats were transferred to the basal semisynthetic diet and were killed 3 weeks later. SAM treatment (384 mumol/kg/day, i.m.), was started 1 week after 1,2-DMH and was continued up to the end of the experiment. Controls received solvent alone. SAM exerted an inhibitory effect on the induction of preneoplastic and neoplastic lesions. For example, nodules with diameters of 1-2 and 2-6 mm exhibited a decrease in both incidence and number per liver, while no such inhibitory effect was seen in the category of larger nodules. Furthermore, hepatocellular carcinoma (HCC) also exhibited a decrease in the SAM-treated group. The number/liver and incidence were 0.04 and 4.8% respectively in the SAM-treated group, compared to 0.38 and 37.8% in the control group. Microscopic examination showed the presence of well-differentiated carcinomas and atypical nodules in control rats, while only one small, well-differentiated tumor and one nodule with patterns of initial transformation were seen in SAM-treated rats. No patchy staining of glutathione-S-transferase, indicative of remodeling, was observed in nodules of both SAM-treated and control rats. Nodules and HCCs developing in SAM-treated rats exhibited a relatively high number of apoptotic bodies. Apoptotic bodies count showed 2.8- and 1.8-fold increases in nodules and HCCs of SAM-treated rats with respect to controls. These results indicate that SAM exerts a chemopreventive effect on hepatocarcinogenesis induced by the OA model. SAM seems to be more effective in inhibiting nodule to HCC progression than on the growth of nodule per se. The inhibitory effect is associated with an increase in cell loss by apoptosis in nodules and HCC.

Lasiocarpine (LC), a pyrrolizidine alkaloid, is able to induce a series of chronic and progressive lesions in rat liver, including a long-lasting block in the cell cycle, the appearance of enlarged hepatocytes (megalocytosis), fibrosis, cirrhosis and malignant neoplasma. In this study the effect of transplantation of normal hepatocytes on the development of LC-induced chronic lesions in rat liver was examined. Two-month-old male Fischer 344 rats were given a single dose of LC (80 mumol/kg i.p.). Four weeks later all animals were subjected to 2/3 partial hepatectomy (PH). In addition, at the time of PH one group of rats were transplanted with normal hepatocytes isolated from a syngeneic donor (10(6) cells/rats via the portal vein), while the other group received only the culture medium. All rats were killed 14 weeks after the operation. Grossly, the liver of rats exposed to LC followed by PH with no transplantation of normal hepatocytes was small in size (% liver wt/body wt 1.66 +/- 0.08) and exhibited a few whitish nodules. Histologically, approximately 88% of the liver section was occupied by enlarged hepatocytes and hepatocyte nodules composed of smaller hepatocytes developed in every animal in this group. In addition, extensive bile ductular proliferation was present. However, the liver of rats that were similarly treated but received normal hepatocytes were significantly larger in size (% liver wt/body wt 2.16 +/- 0.07) and were almost completely free of megalocytosis, bile ductular proliferation and hepatocyte nodules. These findings indicate that transplantation of normal hepatocytes is able to modulate the development of chronic liver lesions induced by LC and may be relevant to the pathogenesis of progressive liver diseases such as neoplasia and cirrhosis.

Feeding excess orotic acid (OA) in the diet promotes the carcinogenic process in different organs including the liver. A number of metabolic and genetic disorders are associated with increased synthesis of endogenous OA and some of these disorders appear to pose an increased risk of liver cancer development. This study therefore examines whether excess OA of endogenous origin also exerts a promoting effect on hepatocarcinogenesis in the mouse and the rat. Increased endogenous synthesis of OA was achieved by (i) feeding a diet deficient in arginine (AD) and (ii) feeding excess dietary carbamylaspartate (CA), a precursor for the synthesis of OA. A single dose of diethylnitrosamine (DENA) was given i.p. to male Fischer 344 rats (200 mg/kg) or to male DBA/2 mice (90 mg/kg). One week later they were placed on either AD diet or the same diet supplemented with 1.35% arginine (AS) for a total of 4 weeks. Two-thirds partial hepatectomy (PH) was performed at the end of the second week. All animals were then transferred to a control semisynthetic basal diet for a total of 20 weeks before they were killed. The results indicated that AD diet increased the incidence of hepatic nodules in both rats (percentage area occupied by nodules was 4.7 +/- 0.4 in the AD group compared to a control value of 0.7 +/- 0.5) and mice (4/10 mice had nodules > 5 mm diameter in the AD group while none in the AS group had such large nodules). In another experiment male Fischer 344 rats similarly initiated with DENA were exposed to either basal diet or basal diet containing 2% CA for 4 weeks coupled with PH performed at the end of the second week. This regimen was followed by 20 weeks of feeding basal diet to both groups. Rats given CA developed larger hepatic foci and nodules (0.84 +/- 0.56 mm3) compared to the control group, which was fed basal diet throughout the experiment (0.07 +/- 0.03 mm3). Further, both AD diet and dietary CA, like dietary OA, induced an increase in hepatic uridine nucleotides. Taken together, these results suggest that increased levels of endogenously synthesized OA, like exogenously supplied excess OA, can induce an imbalance in hepatic nucleotide pools and can exert a promoting effect on hepatocarcinogenesis.

One of the proposed mechanisms by which phenobarbital (PB) promotes hepatocarcinogenesis in the rat is by differential mitoinhibition. However, our earlier studies indicated that PB inhibited DNA synthesis in vitro in hepatocytes isolated from both surrounding non-nodular liver and hepatic nodules promoted by orotic acid (OA). Since nodules generated by one promoter need not necessarily be resistant to another promoter, the present study was undertaken to determine whether foci/nodules promoted by PB itself are resistant to the mitoinhibitory effects of PB. Accordingly, rats were initiated with diethylnitrosamine (DENA, 200 mg/kg i.p) and promoted with PB (0.07% of PB as its sodium salt) in their drinking water for 16 or 33 weeks. In vitro studies indicated that PB (3-5 mM) inhibited DNA synthesis induced by epidermal growth factor (EGF) in hepatocytes from surrounding non-nodular liver as well as from nodules promoted by PB for 33 weeks. In another experiment, initiated rats exposed to PB for 33 weeks were subjected to either two-thirds partial hepatectomy (PH) or sham hepatectomy. Hepatocytes were labelled with tritiated thymidine in vivo for 48 h. Autoradiographic analysis indicated that in the presence of PB, the hepatocytes from both foci/nodules and the surrounding non-nodular liver responded to PH to the same extent. In addition, they both responded to PH less efficiently as compared to the corresponding controls. Further, initiated rats exposed to PB for 16 weeks when subjected to PH and killed 4 weeks thereafter, the percentage area occupied by gamma-glutamyltranspeptidase-positive foci/nodules in the PB group increased, but to the same extent as in initiated control rats not exposed to PB. The above results raised an interesting possibility that the lack of resistance of the PB-promoted nodules to the mitoinhibitory effects of PB may be because the PB-promoted nodule does not express a resistant phenotype. To examine this aspect, the response of hepatocytes from 33 week PB-promoted nodules to the mitoinhibitory effects of OA was examined. The results indicated that OA (60-120 microM) inhibited EGF-induced DNA synthesis in hepatocytes isolated from both nodules as well as from surrounding non-nodular liver. These results suggest that PB is a mitoinhibitor but may not provide a strong differential growth advantage to foci/nodules in response to a proliferative stimulus. Further, the nodules promoted by PB do not appear to express the resistant phenotype, defined as being resistant to the mitoinhibitory effects of OA and PB.

Our earlier studies demonstrated that the beta-hydroxy-beta-methylglutaryl coenzyme A (HMG CoA) reductase gene is hypomethylated and overexpressed in hepatic nodules initiated by 1,2-dimethylhydrazine (1,2-DMH). The study presented here examined whether the pattern of DNA methylation of the HMG CoA reductase gene in hepatic nodules reflected carcinogen-DNA interaction during initiation. Accordingly, hepatic nodules were generated in male Fischer 344 rats with either 1,2-DMH or aristolochic acid (AA), which interact predominantly with the guanine and adenine bases in DNA, respectively. DNA from individual nodules was restricted with HpaII, MspI, and HhaI, and the fragments obtained were hybridized to a cDNA probe for HMG CoA reductase. The results indicated that the hypomethylation pattern in the reductase gene in the nodules initiated with these two carcinogens was similar, although they interacted with different bases in the DNA. The question still remained whether the DNA fragments obtained by digesting the two sets of nodules with the restriction endonucleases were from the same domains in the genome of HMG CoA reductase. To examine this, probes for the different domains of the HMG CoA reductase gene were generated from the cDNA using the restriction enzyme Accl. Three probes were obtained: (i) a 5'-end fragment corresponding to the membrane-spanning region, (ii) a second fragment corresponding to the 3'-end of the protein, and (iii) a third fragment spanning the region between (i) and (ii). Each of these probes was radiolabeled and hybridized to the HpaII- and HhaI-generated fragments from the DNA of hepatic nodules initiated with 1,2-DMH and AA. Irrespective of the carcinogen used for initiation, hypomethylation occurred in all three domains of the gene. More important, the pattern of hypomethylation was similar in the nodules initiated by the two carcinogens. Furthermore, an overall similarity was seen in the hypomethylation patterns in the c-myc and c-Ha-ras genes in the DNA of nodules initiated with either 1,2-DMH or AA. These results raise the possibility that the pattern of hypomethylation established in the hepatic nodules may not directly reflect the interaction between the initiating carcinogen and DNA but may represent a unique phenotype of hepatic nodules.

In the present study, sequential histopathological changes during hepatocarcinogenesis promoted by orotic acid were examined. Male Fischer 344 rats were given 1,2-dimethylhydrazine.2HCl (100 mg/kg, i.p.) 18 h after 2/3 partial hepatectomy. After 1 week of recovery, they were divided into 2 groups; group 1 was continued on a semisynthetic basal diet while the group 2 received the basal diet containing 1% orotic acid. Rats were sacrificed after 5, 10, 20, 29, 40 and 53 weeks of promotion. Histopathological analysis indicated that emergence of hepatocellular carcinomas was preceded first by foci of morphologically and histochemically altered hepatocytes and then by the appearance of hepatocyte nodules. Clear cell foci, eosinophilic ground glass foci and gamma-glutamyltransferase positive foci were detectable after 5 weeks in initiated rats fed orotic acid. Hepatocyte nodules developed in 56% of the rats after 20 weeks of promotion, while the first hepatocellular carcinoma was observed in one rat sacrificed after 29 weeks of orotic acid promotion. Cancer incidence steadily increased with the duration of the orotic acid treatment and 59% developed hepatocellular carcinomas with 30% metastasis to lungs by 53 weeks of promotion. A relevant feature of this model is that during exposure to orotic acid no liver hyperplasia, nor bile duct or oval cell proliferation were seen and the liver architecture in the tissue surrounding focal lesions was well preserved throughout the sequence.

One of the hallmarks of initiated hepatocytes is their resistance to several hepatotoxins. This property forms the basis for their selective growth under conditions which are inhibitory to the non-initiated hepatocytes. Selective growth of initiated hepatocytes also occurs, albeit at a low level, in initiated rat liver without exposure to any known promoting regimen and/or in the absence of any known selective pressure to which initiated hepatocytes can possibly be resistant. This latter phenotypic property of initiated hepatocytes was further characterized by comparing the kinetics of response of hepatocytes in gamma-glutamyl transferase positive foci and in the surrounding liver to 2/3 partial hepatectomy both in the presence and in the absence of a promoting regimen. Male Fischer 344 rats (130-150 g) were initiated with a single dose of diethylnitrosamine and 1 week later they were placed on either a semi-synthetic basal diet or a promoting diet containing 1% orotic acid. Partial hepatectomy was performed 15 weeks after initiation and animals from both groups were killed at 12, 16, 20, 24, 30, 36, 48, 72 or 96 h after operation. Each animal received a pulse of 3H-labelled thymidine 1 h prior to killing. Autoradiographic studies revealed that hepatocytes in gamma-glutamyl transferase positive foci in the livers of rats fed the basal diet were significantly labelled at 16 h post-partial hepatectomy while surrounding hepatocytes were still virtually quiescent (LI 12.7 +/- 4.7 versus 1.2 +/- 0.5%, respectively). Higher labelling index in foci compared to the surrounding liver was also seen at 20 h post-PH (36.9 +/- 2.6 versus 21.5 +/- 2.4). Similar earlier response of hepatocytes in gamma-glutamyl transferase positive foci was also seen in initiated rats exposed to dietary orotic acid. In addition, orotic acid treatment appears to have imposed a slight delay on the entry of hepatocytes in the surrounding liver into 'S' phase and thereby enhancing the differential of growth response between these two populations.

It has been hypothesized that orotic acid (OA) promotes rat liver carcinogenesis by a differential mitoinhibitory mode. Consistent with this hypothesis, hepatic nodules are relatively resistant to OA-induced mitoinhibition. OA-induced mitoinhibition is dependent on the metabolism of OA to uridine nucleotides. The present studies investigate the uptake and metabolic pathway of OA, both in vivo and in vitro, as a possible basis for the resistance of hepatic nodules to OA-induced mitoinhibition. Rats bearing hepatic nodules exposed to 1% dietary OA exhibited increased levels of uridine nucleotides in the surrounding non-nodular liver (from 0.44 to 0.70 mg/g liver) but not in the hepatic nodules. Further, following administration of [3H]OA i.p., nodules have significantly lower levels of acid-soluble radioactivity compared to the non-nodular surrounding tissue. Furthermore, most of the acid-soluble radioactivity was present as uridine nucleotides, suggesting that the OA taken up was converted to uridine nucleotides. Similarly, hepatocytes from nodules in primary culture incubated with radiolabeled OA, have significantly lower levels (46-60%) of acid-soluble radioactivity. These results suggest that the decreased uptake of OA by hepatic nodules may be a factor contributing to the observed resistance of hepatic nodules to the mitoinhibitory effects of OA.

Previous work from this laboratory has revealed that a minimum of 10-20 weeks of continuous exposure to 1% dietary orotic acid (OA) is necessary for this regimen to exert a significant promoting effect on the carcinogenic process in rat liver. The present study investigates the effect of partial hepatectomy (PH), given during a short-term exposure (4 weeks) to OA, on the development of hepatocyte nodules (HN) and hepatocellular carcinoma (HCC) initiated by diethylnitrosamine (DEN). Male Fischer 344 rats (130-150 g) were given a single dose of DEN (200 mg/kg body wt i.p.). Starting a week later they were fed either a semisynthetic basal diet (BD) or the same diet containing 1% OA for 2 weeks; two-thirds PH was then performed followed by another 2 weeks of BD or OA diet respectively. At the end of this treatment some animals from both groups were killed while the rest were continued on BD and killed at 20 or 56 weeks thereafter. The results showed no difference between the two groups in the incidence of gamma-glutamyltransferase-positive foci when rats were killed at 2 weeks after PH. However, 4 week exposure to OA coupled with PH significantly enhanced the incidence of HN and HCC when this protocol was followed by 20 or 56 weeks of BD feeding respectively, leading to 63% incidence of HCC in the OA-fed group, while no HCC was observed in control animals. It is concluded that a type of stable or permanent change(s) ('imprinting' or 'memory effect') is induced in the initiated rat liver by this treatment, which imposes a promoting environment in the liver even after withdrawal of the promoter.

Experiments were conducted to determine whether the excessive orotic aciduria, induced in sparse-fur male mice (spf/Y) deficient in ornithine transcarbamylase (OTC), may be regulated by some inhibitors, such as acivicin (0.014 mmol/100 g body weight, i.p.), N-(phosphonoacetyl)-L-aspartate (PALA, 2.5 mg/100 g body weight, i.p.), adenine (3 g/kg diet) and cycloheximide (0.35 mmol/kg body weight, i.p.). We also administered ornithine (1 mmol/100 g body weight, i.p.), a substrate of the urea cycle, to alleviate the metabolic deficiency of arginine in spf/Y mice which may also be responsible for excessive orotic aciduria. The orotic aciduria remained insensitive to acivicin, indicating mitochondria as the source of carbamyl phosphate. However, orotate excretion was significantly decreased by PALA (P < 0.01), due to its effect on the aspartate transcarbamylase activity. The ingestion of adenine resulted in an increase (P < 0.05) of urinary orotate, suggesting the blockage of the utilization of orotate for nucleotide biosynthesis. Ornithine administration led to a reduction (P < 0.01) of the excretion of orotate induced by the OTC deficiency in these mice, indicating that one of the regulatory steps in its synthesis may be the availability of ornithine. There were no changes in urinary orotate excretion in spf/Y mice when treated with cycloheximide. On the other hand, pretreatment with cycloheximide in an artificial model of OTC deficiency (Swiss-ICR normal mice on an arginine-deficient diet treated thereafter with norvaline, an inhibitor of OTC), caused a significant decrease in urinary orotate. These results suggest that spf/Y mice are unique in that the increased synthesis of orotate is not sensitive to cycloheximide. Perhaps this may reflect an adaptive phenomenon developed by the mutant mice to handle excess mitochondrial carbamyl phosphate and orotic acid.

The present study was designed to determine the mechanism by which orotic acid, a rat liver tumor promoter, inhibits DNA synthesis in normal hepatocytes in primary culture. Our results indicate that orotic acid inhibited the epidermal growth factor induced expression (mRNA) of both M1 and M2 subunits of ribonucleotide reductase while the expression of c-fos, c-myc, c-Ha-ras and beta-actin was not inhibited to any significant extent. These studies suggest that ribonucleotide reductase may be one target for orotic acid-induced mitoinhibition.

The present study was designed to determine the long-term effects of orotic acid (OA), a multi-organ tumor promoter, in rats not exposed to any carcinogen. Male Fischer 344 rats (130-150 g) were divided into two groups and given either a semisynthetic basal diet (BD) or the same diet containing 1% OA. Animals from both groups were killed after 1 or 2 years of treatment. Foci of placental glutathione-S-transferase (GST 7-7) positive hepatocytes were observed in the livers of both BD and OA fed rats killed after 1 year. However, they were more in number in animals receiving OA (156 +/- 21 versus 51 +/- 11/cm3). After 2 years, hepatic nodules were seen in almost all the animals given OA and in approximately 30% of the rats given BD. The nodules were of two main types: (i) a reddish-brown type, present in 85% of rats exposed to OA and in 27% of rats given BD, and (ii) a greyish-white type, found in 50% of animals fed OA and in none of the animals fed BD. These two types of lesions were also histologically different. Reddish-brown nodules were composed of slightly enlarged hepatocytes resembling normal surrounding tissue, while greyish-white nodules were similar in structure and are indistinguishable from hepatic nodules induced by genotoxic chemical carcinogens. The results are interpreted to suggest that the foci/nodules seen in OA-fed rats are due to a promoting effect of OA on spontaneously arising and/or diet-induced altered cells.

Our earlier studies indicated that orotic acid, a precursor for pyrimidine nucleotide biosynthesis, exerts a promoting effect on rat hepatocarcinogenesis. The present study was designed to determine the optimum conditions of exposure to orotic acid required for promotion of hepatocarcinogenesis in the initiated rats. The first series of experiments was designed to determine the optimum dose of orotic acid needed to exert its liver tumor promoting effect. Accordingly male Fischer rats were given diethylnitrosamine (200 mg/kg, i.p.) or 0.9% NaCl. One week later carcinogen-injected rats were divided into six groups and fed either basal diet or the same diet containing 0.1, 0.5, 1, 2 or 4% orotic acid. Rats given 0.9% NaCl were fed 4% orotic acid. Two-thirds partial hepatectomy was performed on all animals 10 weeks after starting on their respective diets, and all groups were killed 3 weeks thereafter. Analysis of gamma-glutamyltransferase-positive foci and nodules revealed that 0.5-1% orotic acid in the diet is sufficient to exert a significant promoting effect on the selective growth of initiated hepatocytes, while higher concentrations of orotic acid were only marginally more effective. No gamma-glutamyltransferase-positive foci were observed in animals given 4% orotic acid diet following saline injection. Using 1% orotic acid as the promoting regimen, in the next series, the minimum exposure time required for dietary orotic acid to promote liver carcinogenesis was determined. Male Fischer 344 rats were given i.p. either 1,2-dimethylhydrazine dihydrochloride (100 mg/kg) or 0.9% NaCl 18 h after 2/3 partial hepatectomy. After 1 week of recovery one group of rats was continued on a semisynthetic basal diet, while others were transferred to the same basal diet containing 1% orotic acid. Rats that were on the 1% orotic acid diet were progressively transferred to the basal diet after 5, 10, 20, 29 and 40 weeks of exposure. All rats were sacrificed 54 weeks after the beginning of the experiment. The results indicate that 100% of the initiated rats developed hepatic nodules whether or not they were exposed to an orotic acid-containing diet. However, the incidence of hepatocellular carcinoma was greatly increased in animals exposed to the orotic acid diet, with 42% incidence in initiated rats given orotic acid diet for 10 weeks and up to 75% in those exposed to this diet for 40 weeks. Further, promotion by orotic acid exhibited a high metastatic potential with 33-60% metastasis to the lungs.(ABSTRACT TRUNCATED AT 400 WORDS)

The present study was designed to determine whether a delay in the start of the promoting regimen after the administration of a carcinogen would influence the promoting efficacy of orotic acid on the development of hepatocellular carcinoma in rats. Male Fischer 344 rats weighing 130-150 g were injected with a single dose of diethylnitrosamine (200 mg/kg body wt i.p.) then divided into 3 groups: groups 1 and 2 were given semi-synthetic basal diet or the same diet containing 1% orotic acid (OA) respectively starting 1 week after the carcinogen; group 3 received the OA diet starting 5 weeks after the administration of diethylnitrosamine. Animals from these 3 groups were sacrificed after 25, 32, 42 and 60 weeks of being fed their respective diets. The results indicated that delaying the start of the OA diet after the carcinogen resulted in about a 50% decrease in the incidence of hepatic nodules and/or hepatocellular carcinomas at various time points during the experiment. This decrease in promoting efficacy of OA was not apparently explainable by lack of metabolic effects of OA, at least in terms of induction of nucleotide pool imbalance, a condition that appears to be important for OA to exert its tumor promoting effects.

Aristolochic acid (AA), used as an anti-inflammatory agent in the past, is known to be mutagenic and carcinogenic to several organs of the rat, including forestomach, renal pelvis and urinary bladder. However, despite the induction of DNA adducts in the liver, no carcinogenic potential of AA has been reported in the latter organ. The present study was based on the rationale that the lack of carcinogenicity of AA to the liver could be because this chemical may not be necrogenic at the doses examined and liver cell proliferation has been established as an essential component for initiation of liver carcinogenesis in the rat. The results indicated that AA is non-necrogenic to the rat liver. However, a single non-necrogenic dose of AA (10 mg/kg b.w., i.p.) given 18 hours after 2/3 partial hepatectomy initiated liver cell carcinogenesis. The initiated cells are promotable with 1% dietary orotic acid, a liver tumor promoter, to form glutathione-S-transferase 7-7 positive hepatic foci and nodules.

The steady-state levels of c-fos, c-jun and c-myc messenger RNA were investigated in rat liver tissue after proliferative stimuli of different nature-namely, compensatory regeneration induced by partial hepatectomy or carbon tetrachloride administration-and direct hyperplasia induced by four different hepatomitogens: lead nitrate, ethylene dibromide, cyproterone acetate and nafenopin. We show here that whereas c-fos and c-jun expression increased soon after partial hepatectomy or carbon tetrachloride administration, an increased expression of c-jun in the absence of c-fos expression occurred during direct hyperplasia induced by lead nitrate and ethylene dibromide. When hyperplasia was induced by cyproterone acetate and nafenopin, the mitogenic response of the liver was not associated with an increased expression of c-jun or c-fos, despite the fact that the timing of the cell cycle was similar to that observed after partial hepatectomy. Finally, when c-myc expression was analyzed, it was found that proliferative conditions associated with an increased expression of this gene were characterized by an increased expression of c-jun. On the contrary, the hyperplasia induced by cyproterone acetate and nafenopin, which is characterized by a lack of increase in the expression of c-fos and c-jun, was also not associated with an increased c-myc expression. Similar results were obtained in these experiments with the mitogen nafenopin, a peroxisome proliferator. In fact, liver hyperplasia induced by this compound was not preceded or accompanied by an increased expression of c-fos and c-myc. This study suggests that depending on the nature of the proliferative stimulus, an increased expression of c-fos, c-jun and c-myc may not be necessary for in vivo induction of liver cell proliferation.

The possible mechanism(s) responsible for the different effects exerted by proliferative stimuli of different nature on the appearance of enzyme-altered hepatic foci, were investigated in male Wistar rats. Rats given an initiating dose of diethylnitrosamine (150 mg/kg body weight) were fed a diet containing 0.03% acetylaminofluorene for 2 weeks. Between the first and the second week, cell proliferation was induced by a proliferative stimulus of compensatory type (partial hepatectomy) or by a direct mitogenic stimulus (lead nitrate, 100 mumol/kg). The effect of the two different proliferative stimuli on the appearance of gamma-glutamyl transferase-positive foci was monitored by killing the rats for examination at 1, 2, 3, 5, and 6 days after the induction of cell proliferation. The results indicate that while enzyme-altered hepatocytes can be observed as early as 3 days after partial hepatectomy and are characterized by a rapid growth, direct hyperplasia did not exert any effect on the growth capacity of initiated cells. No effect of lead nitrate-induced hyperplasia was observed following three administrations of the mitogen. When platelet-poor plasma taken from animals exposed to the different proliferative stimuli was tested in primary cultures of hepatocytes, it was found that it induced a significant increase in the labeling index of normal hepatocytes. However, while serum taken 6 days after partial hepatectomy was still able to induce a significant increase in the labeling index, platelet-poor plasma from lead-treated rats had lost part of its effect at 5 days after treatment. The inability of direct hyperplasia to stimulate the development of enzyme-altered hepatic foci was not unique to lead nitrate since the same phenomenon was observed when three other hepatomitogens, nafenopin, cyproterone acetate, and ethylene dibromide, were used.

One of the proposed mechanisms by which orotic acid (OA) promotes liver carcinogenesis is by differentially mito-inhibiting the normal hepatocytes while permitting the initiated ones to respond to growth stimuli to form foci/nodules. In an attempt to examine this hypothesis, the present study was designed to determine (i) whether OA inhibits DNA synthesis in normal hepatocytes in vivo, and (ii) whether hepatocytes from hepatic foci/nodules are relatively resistant to the mito-inhibitory effects of OA. The results of this study indicate that OA given i.p. as a tablet of 300 mg at the time of partial hepatectomy (PH) almost completely inhibited liver DNA synthesis. Three days later--a time period by which the implanted tablet disappeared--the hepatocytes resumed DNA synthesis. Exposure to OA results in an accumulation of uridine nucleotides and a decrease in adenosine nucleotides. Creation of such an imbalance in nucleotide pools appears to be important for OA to inhibit DNA synthesis. Adenine (a tablet of 300 mg), an agent that inhibits the metabolism of OA to uridine nucleotides, counteracted the mito-inhibitory effects of OA. To determine whether the hepatocytes in foci/nodules are resistant to the mito-inhibitory effects of OA, rats were initiated with diethylnitrosamine (DENA; 150 mg/kg) and promoted by the resistant-hepatocyte model. Fourteen weeks after the administration of DENA, the rats were subjected to PH in the presence of absence of OA (300 mg tablet). The results indicated that, in contrast to hepatocytes in normal or surrounding non-nodular liver, a subpopulation of hepatocyte foci/nodules appear to be relatively resistant to the mito-inhibitory effects of OA. These findings support the hypothesis that differential mito-inhibition is a possible component in the promoting effect of OA. However, whether this is the mechanism by which OA promotes liver carcinogenesis needs to be further investigated.

This study was designed to determine whether hepatocytes from hepatic nodules are resistant to the mitoinhibitory effects of orotic acid. Hepatic nodules were initiated in Fischer 344 male rats with 1,2-dimethylhydrazine.2HCl (100 mg/kg ip) given 16 hr after two thirds partial hepatectomy and promoted by feeding a diet containing 1% orotic acid. Eight to 9 months later, when persistent nodules had developed, the rats were taken off the orotic acid diet and maintained on a semisynthetic basal diet for 2 to 5 weeks. The effect of orotic acid on the DNA synthesis in the hepatocytes isolated from hepatic nodules and from the surrounding nonnodular liver and from the age- and sex-matched control rats was studied. The results indicated that a dose of orotic acid (120 microM) that almost completely inhibited the transforming growth factor-alpha-induced DNA synthesis in hepatocytes from nonnodular surrounding liver and from age- and and sex-matched control liver could not inhibit the DNA synthesis in hepatocytes from hepatic nodules. These results are consistent with the postulate that orotic acid may promote liver carcinogenesis by a differential mitoinhibition of normal hepatocytes while permitting the initiated hepatocytes to respond to growth stimuli and form hepatic nodules. However, it needs to be determined whether differential mitoinhibition of normal hepatocytes is the mechanism by which orotic acid promotes liver carcinogenesis.

D-Galactosamine is a known hepatotoxin which induces liver cell necrosis via depletion of UTP and other uridine nucleotides. Our previous work indicated that nodular hepatocytes have higher levels of total uridine nucleotides compared to normal liver, and in the present study we investigate the effect of galactosamine treatment on hepatocyte nodules and surrounding liver. Hepatic nodules were generated in male Wistar rats according to the Solt and Farber protocol. Six months after initiation animals received a single injection of D-galactosamine (500 mg/kg i.p.) and were then killed 1, 2, 4 or 7 days later. Histological analysis of liver revealed the presence of extensive liver cell necrosis in normal tissue 1 and 2 days after galactosamine treatment. However, very little or no necrosis was detectable inside hepatic nodules at any time point, indicating that these focal areas are resistant to the cytotoxic effect of galactosamine. This type of resistance could be the expression of a new component in the resistant phenotype of hepatic nodules.

One of the many hypotheses put forward to explain the mechanism by which phenobarbital (PB) promotes hepatocarcinogenesis is by differential mitoinhibition of surrounding hepatocytes while allowing the initiated hepatocytes to respond to growth stimuli and form foci and nodules. Given the similarity in structures between PB and orotic acid (OA), another rat liver tumor promoter, the present investigation was designed to determine (i) whether PB, like OA, exerts its mitoinhibitory effect at a site beyond the growth factor receptor and receptor mediated early events; and (ii) whether PB exerts a differential mitoinhibitory effect by selectively inhibiting the non-initiated hepatocytes but not the initiated hepatocytes in vitro. Our studies demonstrate that, like OA, PB also inhibits DNA synthesis in hepatocytes from normal rat liver in a dose dependent manner with 80-90% at a dose of 6 mM. One target site may lie beyond the growth factor receptor mediated early events because PB inhibited DNA synthesis in hepatocytes primed with the growth factor 24 h earlier. Interestingly, PB inhibited DNA synthesis not only in hepatocytes from non-nodular surrounding liver but also in hepatocytes from persistent hepatic nodules initiated with 1,2-dimethylhydrazine and promoted with OA. Therefore, our results suggest that although PB is a mitoinhibitor of DNA synthesis in hepatocytes, it does not appear to create as strong a differential mitoinhibition between non-nodular surrounding and initiated hepatocytes as is evident in the resistant hepatocyte and OA models. These results raise the question whether differential mitoinhibition is the major contributing factor in the PB mediated rat liver tumor promotion.

Sparse fur hemizygous male mice are over 90% deficient in ornithine transcarbamylase and exhibit increased synthesis of orotic acid. Because our earlier studies have demonstrated that orotic acid is a liver tumor promoter in the rat, it was of interest to determine whether this genetic disorder also increases the risk of tumor promotion. The results revealed that the livers of mutant mice showed a fourfold increase in uridine nucleotides and a 50% decrease in adenosine nucleotides compared to corresponding controls, a pattern of nucleotide pool imbalance similar to that seen in the livers of rats exposed to orotic acid under promoting conditions. Creation of such an imbalance appears to be important for orotic acid to exert its promotional effects. Sparse fur mutant mouse may, therefore, be an ideal animal model to study the tumor-promoting effects of orotate.

This study was designed to determine the possible mechanism by which orotic acid exerts its mitoinhibitory effect on rat hepatocytes in primary culture. Orotic acid inhibited, dose-dependently DNA synthesis in hepatocytes induced by epidermal growth factor, transforming growth factor alpha, hepatocyte growth factor, acidic fibroblast growth factor, or plasma from rats exposed to various liver cell-proliferative stimuli, such as two-thirds partial hepatectomy, lead nitrate, cyproterone acetate, ethylene dibromide, or a diet deficient in choline. Further, orotic acid inhibited DNA synthesis even when added 24 h after the hepatocytes were primed with transforming growth factor alpha. Taken together, these results suggested that the target site may not be at the level of the growth-factor receptor and receptor-mediated early events. In a preliminary experiment, orotic acid inhibited the expression of the ribonucleoside diphosphate reductase gene. Exposure to orotic acid results in an imbalance in nucleotide pools characterized by an increase in uridine nucleotides and a decrease in adenosine nucleotides. It is hypothesized that this imbalance in nucleotide pools inhibits the expression of the ribonucleoside diphosphate reductase gene and, therefore, is a likely target for the mitoinhibitory effect of orotic acid.

Our earlier studies had demonstrated that inhibition of DNA methylation following carcinogen treatment potentiated initiation of the carcinogenic process in the rat liver system. The hepatic nodules developed by initiation-promotion protocols showed a characteristic hypomethylation in the cell-cycle-related genes c-fos, c-myc and c-Ha-ras. In the present study we have found that the gene for beta-hydroxy-beta-methyl glutaryl coenzyme A reductase, a major rate-limiting enzyme in the biogenesis of mevalonate, is also hypomethylated at both CCGG and GCGC sites and expressed in hepatic nodules. This gene, however, did not exhibit hypomethylation in CCGG sequences in non-nodular surrounding liver, livers from rats subjected to two-thirds partial hepatectomy, or exposed to initiator alone (1,2-dimethylhydrazine given 18 h after partial hepatectomy) or to diets containing 1% orotic acid alone (promoting regimen). The activity of the enzyme and mevalonate formation are positively correlated with DNA synthesis and cell proliferation--two key components of the carcinogenic process. Taken together, the results suggest that hypomethylation of specific genes occurs in the carcinogenic process and this altered pattern of DNA methylation may play a role in the growth of the nodules.