Introduction
============

Carcinoma of the ampulla of Vater is an infrequent neoplasia. Histologically the tumour is an adenocarcinoma arising from either pancreatic ducts, acinus or bile ducts and is often of a low grade malignancy. Both sexes are affected, the male to female ratio being 2:1 with most patients being between 50 and 69 years of age \[[@B1],[@B2]\]. Early diagnosis is vital because after a complete surgical resection the five-year survival rate can be expected to be approximately 40% \[[@B3]\].

Early symptoms (pain, cholestasis, intermittent jaundice) result from the extension of the tumour and either the obstruction or the compression of the papillary duct. Extension of the tumour into the duodenum causes occult anemia or intestinal obstruction \[[@B2],[@B4]\].

Endoscopic Ultrasonography (Endosonography, EUS) is a minimally invasive new imaging procedure. In this method a high frequency transducer (7.5 and 12 MHz) is placed into the duodenum, offering a detailed ultrasound image of the duodenal wall, at the convergence of the common bile and pancreatic duct. Even small papillary tumour masses can be detected with this method \[[@B5]\].

In this prospective study we evaluate the accuracy of EUS in detection and preoperative TNM staging (T and N classification) of ampullary carcinomas. Data on clinical presentation, endoscopic findings, endosonographic findings, US and CT scan findings, treatment and follow-up of 20 patients with carcinoma of the ampulla of Vater are reported.

Patients and Methods
====================

Between July 1993 and February 2000 twenty patients having carcinoma of the ampulla of Vater were prospectively evaluated. The study was approved by the Ethics Committee of the Hospital. An Informed consent for diagnostic examinations was obtained from all patients. Seven women and 13 men, aged 40 to 87 years with a median age of 67 years were included in the study. ERCP and clinical findings of a subgroup of these patients were in part reported elsewhere \[[@B6]\].

The diagnosis was confirmed by histological examination of the specimens, taken at either endoscopy or surgery. All patients were subjected to clinical and laboratory tests. All patients were examined by Ultrasonography (US) and CT scan prior to endoscopic examination. Conventional CT scans were performed with a Philips Lx scanner with a slice thickness of a 10 mm, with oral and intravenous contrast in 13 patients (those diagnosed prior to 1997). Seven patients were subjected to spiral CT (after 1997).

Detection of a tumour mass by means of US and CT scan with direct (visualization of the tumour) or indirect signs of tumour (bile or pancreatic duct dilatation, presence of lymph nodes), and distal metastasis were noted.

Endosonographic examination was also performed in all patients. The Olympus Co GF-EUM3 system (frequency: 7.5 or 12 MHz) with a water filled balloon technique was used. Size and echogeneity of the tumour, local invasion, existence of suspected metastatic lymph nodes and anatomical relation of the tumour to the adjacent abdominal structures were assessed.

Lymph nodes were assessed for malignancy on the basis of four well established criteria which were: larger than 10 mm, round, homogeneous echo pattern and sharp borders \[[@B7]-[@B10]\]. T and N classification, according to the TNM staging system \[[@B11]\] was also recorded. (Table [1](#T1){ref-type="table"}).

::: {#T1 .table-wrap}
::: {.caption}
######

T N M classification of carcinoma of the ampulla of Vater
:::

  **T (Primary tumour)**
  ------------------------------ -----------------------------------------------------------------------------------------------------------------------------------------
  **Tx**                         Primary tumour cannot be assessed
  **T1**                         Tumour limited to the ampulla of Vater or aphincter of Oddi
  **T2**                         Tumour invades duodenal wall
  **T3**                         Tumor invades 2 cm or less into pancreas
  **T4**                         Tumor invades more than 2 cm into pancreas and/or into other adjacent organs

  **N (Regional lymph nodes)**

  **Nx**                         Regional lymph nodes cannot be assessed
  **NO**                         No regional lymph nodes metastasis
  **N1**                         Regional lymph nodes metastasis

  **M (Distant metastasis)**

  **Mx**                         Distant metastasis not assessable
  **M0**                         No distant metastasis
  **M1**                         Distant metastasis: Hepatic metastasis, peritoneal dissemination, lymph nodes metastasis along the splenic vein or at the splenic hilum
:::

Duodenoscopy and ERCP were performed in all patients after US, CT and EUS assessment. Endoscopic biopsies were taken after sphincterotomy. Seventeen patients were operated upon. Patients were followed-up for a period of 2--75 months (median 27 months) until February 2000. One patient refused any therapeutic procedure and was lost to follow-up one month after diagnosis.

Statistical analysis was done by the chi-square paired sample test for proportions with Yates continuity correction. Survival analysis in 17 patients that underwent surgery was performed using the Kaplan-Meier procedure.

Results
=======

Symptoms, clinical characteristics and laboratory tests of all patients are shown in table [2](#T2){ref-type="table"}. The most common symptoms were jaundice (70%), weight loss (55%), epigastric pain (50%), fever (45%), nausea and/or vomiting (35%). An elevated Alkaline Phosphatase (SAP) and γ-Glutamyl Transpeptidase (95%) were the most common laboratory abnormalities. Elevated ALT and AST (40%) and anemia (35%) were also common. In table [3](#T3){ref-type="table"} we summarize endoscopic, imaging, endosonographic findings as well as outcome of all patients.

::: {#T2 .table-wrap}
::: {.caption}
######

Clinical and Laboratory data of the patients on admission. Anemia is defined as a decrease of haemoglobin more than 10% of the normal value. Elevated levels of SAP and γ-Gt (Cholestatic enzymes) and ALT, AST are defined as more than double of the normal values.
:::

  Case   Sex   Age   Jaundice   Fever   Pain   Nausea   Weight loss   Anemia   SAP, γ Gt   ALT, AST
  ------ ----- ----- ---------- ------- ------ -------- ------------- -------- ----------- ----------
  1      M     64    \+         \-      \+     \+       \+            \+       \+          \+
  2      M     43    \+         \+      \+     \-       \+            \+       \+          \+
  3      M     70    \+         \-      \-     \-       \+            \+       \+          \+
  4      M     80    \+         \-      \-     \-       \+            \+       \+          \+
  5      M     67    \+         \-      \-     \-       \-            \-       \+          \-
  6      F     75    \+         \+      \+     \-       \+            \-       \+          \-
  7      F     47    \+         \+      \+     \-       \-            \-       \+          \-
  8      M     62    \-         \+      \+     \+       \-            \-       \+          \-
  9      M     57    \+         \+      \-     \-       \+            \-       \+          \-
  10     F     78    \+         \+             \+       \+            \+       \+          \+
  11     M     40    \-         \-      \-     \-       \-            \-       \+          \-
  12     F     64    \-         \-      \-     \-       \-            \-       \+          \-
  13     M     77    \-         \-      \+     \+       \-            \-       \-          \-
  14     M     87    \+         \+      \+     \+       \+            \-       \+          \-
  15     F     68    \+         \+      \+     \+       \-            \-       \+          \-
  16     M     73    \-         \-      \-     \-       \-            \-       \+          \-
  17     F     63    \-         \-      \+     \-       \-            \-       \+          \-
  18     F     59    \+         \-      \+     \+       \+            \-       \+          \+
  19     M     69    \+         \+      \-     \-       \+            \+       \+          \+
  20     M     72    \+         \-      \-     \-       \+            \+       \+          \+
:::

::: {#T3 .table-wrap}
::: {.caption}
######

Endoscopic biopsy, US and CT scan findings (direct or indirect signs), EUS classification for T and N (TNM staging), treatment, follow-up and outcome of the 20 patients with cancer of the ampulla of Vater.
:::

  Case   Endoscopic biopsy   US         CT         EUS T and N   Treatment        Follow-up (Febr 00)   Outcome (Febr 00)
  ------ ------------------- ---------- ---------- ------------- ---------------- --------------------- -------------------
  1      Ca                  Indirect   Direct     T3N1          Palliative       43                    Died
  2      Ca                  Indirect   Indirect   T2N1          Whipple          64                    Died
  3      \-                  Indirect   Indirect   T3N1          Local excision   2                     Died
  4      Ca                  Indirect   Indirect   T2N0          Stent            63                    Died
  5      \-                  Direct     Indirect   T3N1          Lost             lost                  Lost
  6      normal              Indirect   Indirect   T3N1          Whipple          75                    Died
  7      Ca                  Indirect   Indirect   T4N1          Local excision   34                    Alive
  8      Ca                  Indirect   Indirect   T2N1          Whipple          21                    Alive
  9      \-                  Indirect   Indirect   T2N0          Stent            23                    Alive
  10     normal              Indirect   Indirect   T3N1          Palliative       7                     Died
  11     Ca                  Direct     Indirect   T2N1          Whipple          30                    Died
  12     Normal              Indirect   Indirect   T3N1          Local excision   26                    Alive
  13     Ca                  Indirect   Indirect   T3N1          Whipple          23                    Alive
  14     Ca                  Indirect   Indirect   T4N1          Whipple          2                     Died
  15     Ca                  Indirect   Direct     T3N1          Local excision   39                    Died
  16     Ca                  Indirect   Direct     T2N1          Whipple          22                    Alive
  17     Dysplasia           Indirect   Indirect   T2N0          Whipple          15                    Alive
  18     Ca                  Indirect   Direct     T2N1          Whipple          12                    Alive
  19     Ca                  Direct     Indirect   T3N1          Whipple          8                     Alive
  20     Ca                  Indirect   Indirect   T3N1          Whipple          3                     Alive
:::

The endoscopic appearance of the papilla was abnormal (polypoid or exophytic tumour mass) in 17/20 patients (Fig. [1](#F1){ref-type="fig"}, Fig [3](#F3){ref-type="fig"}). In three cases the tumour was evident only after endoscopic sphincterotomy of a protruding papilla. In the rest of the cases a tumour mass was evident. On ERCP a dilatation of the peripheral part of the common bile duct (15/20) and of the main pancreatic duct (6/20) were found. Multiple endoscopic biopsies were taken from 17 patients. In one patient endoscopic biopsies showed only severe dysplasia, while in three patients the biopsies were normal. Carcinoma of the ampulla of Vater was diagnosed in the remaining 13 patients.

![Polypoid, exophytic mass of the ampulla of Vater. Endoscopic appearance (**T**: tumour mass).](1471-2482-2-1-1){#F1}

![Polypoid tumour mass of the papilla of Vater with ulcerations. Tumour partially obstructs the enteric lumen (**T**: tumour mass).](1471-2482-2-1-3){#F3}

Three patients had a surgical excision based on EUS findings despite an almost normal endoscopic appearance of the ampulla. Histology of the surgical specimens, comfirmed the presence of a carcinoma.

On conventional US, the tumour was identified only in 3 cases (15%) as a hypoechogenic mass at the peripheral part of the common bile duct. In 14 patients indirect signs of malignancy (dilatation of the common bile duct: 12/20, main pancreatic duct dilatation: 2/20, enlarged lymph nodes: 9/20), were noted. In two patients enlarged lymph nodes along the splenic vein were detected (Ml according to the TNM classification). In the remaining three patients the examination was completely normal.

On CT scan, findings were suggestive of a malignant tumour at the distal part of the common bile duct in 4 cases. 2/13 in conventional and 2/7 in spiral CT scan (30%) with an overall accuracy of 20%. Indirect signs of malignancy were evident in 16/20 (80%) cases (dilatation of the common bile duct: 14/20, main pancreatic duct dilatation: 5/20, enlarged lymph nodes: 12/20). In two patients enlarged lymph nodes at the splenic hilum and along the splenic vessels were detected (Ml). In one patient peritoneal dissemination of the mass was shown (Ml).

On EUS tumour was identified in all patients. A heterogeneous hypoechoic mass was the common presentation (fig [2](#F2){ref-type="fig"}, fig [4](#F4){ref-type="fig"}). T and N classification was feasible in all cases. Invasion of the duodenal wall (T2) was found in 8 patients. Tumour invasion within 2 cm on the pancreatic parenchyma was found in 10 patients. These patients were classified as having a T3 carcinoma. Two patients had a tumour that invaded more than 2 cm into the pancreas and they were classified as T4. In 17 patients suspected metastatic lymph nodes were detected (N1).

![Corresponding endosonographic image (7.5 MHz) of the same (fig. [1](#F1){ref-type="fig"}) patient. Hypoechoic mass invading the duodenal wall. Carcinoma of the ampulla of Vater:**T2N0**. (**DL:** duodenal lumen, **T:** tumour mass, **CBD:** common bile duct, **m:** muscularis propria, **nLN:** non metastatic lymph node, **P:** pancreas).](1471-2482-2-1-2){#F2}

![Corresponding endosonographic image (7.5 MHz) of the same (fig. [3](#F3){ref-type="fig"}) patient. Hypoechoic mass invading the duodenum. Enlarged hypoechoic regional lymph nodes are also present. Carcinoma of the ampulla of Vater:**T3N1**. (**DL:** duodenal lumen, **T:** tumour mass, **LN:** metastatic lymph nodes).](1471-2482-2-1-4){#F4}

Whipple resection (pancreatoduodenectomy) was performed in 11 patients. Four patients had a local resection of the tumour, 2 patients had a palliative anastomosis. In total 17 patients were operated upon. Two patients refused surgery and had a stent endoscopically placed.

Table [4](#T4){ref-type="table"} shows the findings of US, CT and EUS concerning tumour and LN staging as well as indirect signs of tumour presence compared to the surgical findings. Preoperative T staging by means of EUS was confirmed in 14/17 patients (82%). From the remaining 3 patients, in 2 the T stage was overestimated (T3 and T4 instead of T2 and T3 respectively) and in one underestimated (T2 instead of T3). N classification was surgically confirmed in 12/17 (71%) of the cases. All of the remaining 5 patients were overestimated by means of EUS. No metastatic lymph nodes were found in surgical samples.

::: {#T4 .table-wrap}
::: {.caption}
######

Evaluation of ampullary cancer by means of US, CT scan and EUS and comparison to surgery findings in 17 patients who underwent surgery. Distribution of T stage, enlarged lymph nodes (for US and CT scan), N stage (for EUS and Surgery), Common bile duct (CBD) and main pancreatic duct (MPD) dilatation (for US and CT scan) and M stage (for US, CT scan and Surgery).
:::

                                **US**                           **CT scan**                      **EUS**                       **Surgery**
  ----------------------------- -------------------------------- -------------------------------- ----------------------------- ---------------------------
  **T stage^\*^**               Advanced 2/17 (12%)              Advanced 4/17 (24%)              Early: 6/17 Advanced: 11/17   Early: 8/17
  **Enlarged LN or N stage**    8/17 (47%)                       11/17 (65%)
  **M stage**                   2/17                             3/17                             NA^\*\*^                      3/17
  **CBD and MPD dialatation**   CBD 12/17 (71%) MPD 2/17 (12%)   CBD 13/17 (76%) MPD 5/17 (29%)   NA                            NA

^\*^ Early stage: T1 and T2 ^\*^ Advanced stage: T3 and T4 ^\*\*^Nonapplicable
:::

Overall accuracy of the EUS for detection of the tumour was 100%. EUS was significantly better than both CT scan (20%) and US (15%) in directly detecting ampullary tumour (p \< 0.001).

The median survival time for the patients who underwent surgery was 64 months (95% CI: 9--119) The Kaplan-Meier survival curve of those operated on is shown in figure [5](#F5){ref-type="fig"}.

Discussion
==========

Cancer of the ampulla of Vater must be differentiated from cancer of the head of the pancreas. Prognosis and potential therapeutic intervention are different. 87% of patients with carcinoma of the ampulla and 47% of those with a malignancy of the duodenum have a potentially operable tumour compared with only 22% of patients with tumours arising from the head of the pancreas \[[@B2],[@B12]\]. Therefore a noninvasive accurate technique for detection and staging is mandatory for these patients.

The endoscopic appearance of the papilla was abnormal in most of our patients. However the distinction between an impacted stone, a benign tumour or a malignancy is not always feasible. ERCP with endoscopic sphincterotomy was a helpful diagnostic procedure in cases of intraampullary tumors with a protruding papilla. In 3 cases of our patients a polypoid mass was evident only after sphincterotomy. However, ERCP is an invasive procedure with a considerable morbidity.

Moreover, endoscopic biopsies do not always allow for reliable diagnosis and preoperative assessment of tumours of the papilla of Vater. Villous adenomas can be associated with an adenocarcinoma and a histological diagnosis of an adenoma does not exclude the presence of an adenocarcinoma elsewhere in the tumour. \[[@B13]-[@B16]\]. In our patients endoscopic biopsies were diagnostic for an adenocarcinoma in 13/17 cases (76%). In one patient a severe dysplasia was reported and in 3 cases histology was completely normal. In other series the positive histological yield was only 50% of the cases \[[@B17]\]. Our results are similar to the studies from Japan and Boston where approximately one-third of the patients were misdiagnosed \[[@B18],[@B19]\].

Staging of the disease is also very important since there is a close correlation between staging and survival rate \[[@B2],[@B20]-[@B22]\]. Conventional imaging techniques such as US and CT scan are not very efficient in both diagnosis and staging. CT scan cannot be used as the only diagnostic procedure for confirming or excluding the diagnosis of ampullary cancer. Staging of ampullary cancer by CT alone was found inaccurate for the evaluation of tumour resectability \[[@B23]\]. Moreover these methods can not detect small tumours. They can indicate the presence of a tumour by indirect signs such a dilatation of the common bile and main pancreatic duct, or the presence of malignant lymph nodes or distal metastases. ERCP can also demonstrate pancreatic and bile duct dilatation \[[@B2],[@B6]\]. In a recent study, CT scan was reported to detect dilatations of peripancreatic veins with nonvisualization of inferior peripancreatic veins as an indirect sign of tumour invasion of the peripancreatic tissue \[[@B24]\].

In our patients conventional US showed a tumour in only 15% of the cases. In 70% of the cases indirect signs of the disease and in 15% no abnormalities were identified. On CT scan, a tumour was directly visualized in only 20% of the patients. In most cases (16/20), indirect signs of the disease were evident but not diagnostic. Conventional CT scan was directly diagnostic in less cases (16%) than spiral CT (30%). This fact underlines the higher accuracy of spiral CT in detecting tumours of the pancreas region \[[@B25],[@B26]\].

EUS provides an accurate method for detecting and staging of ampullary tumours, especially those infiltrating into the pancreas. It also detects tumour infiltration of the proximal blood vessels. However, EUS is unable to detect distal metastasis. Finally, EUS is useful for planning an appropriate therapeutic approach and for preventing unnecessary extensive surgery. EUS findings have been found to correlate well with prognosis \[[@B27]-[@B32]\].

In our study survival for those patients selected by means of EUS for a Wipple\'s procedure was considerably better compared to those who had only a palliative procedure, even if this was not statistically significant due to the small number of patients. This finding agrees with a previous observation according to which EUS for selecting patients for local resection may be a cost-effective strategy \[[@B33]\].

In our patients EUS was highly accurate in directly detecting ampullary tumours making this modality the imaging method of choice. Moreover, in our study the EUS accuracy for tumour invasion (T staging), was 82%. Endosonography had also a relatively high accuracy (71%) in diagnosing regional lymph node metastases (N1 stage).

In agreement with other studies comparing the accuracy of EUS in differentiating between early and advanced ampullary carcinomas (T1/T2 vs T3/T4) where a concordance between 78% and 84% was reported, we found similar accuracy rates \[[@B25],[@B27]-[@B29],[@B31],[@B33]\].

The reported accuracy of EUS in predicting regional lymph nodes metastasis in ampullary carcinoma varies between 59% and 68% \[[@B25],[@B27],[@B34]\]. These findings are comparable or slightly lower than ours (71%). The differences that occur between our results and some of the other studies, could be attributed to more advanced tumours in our patients compared to the mentioned studies. In fact, 60% of our patients had an advanced carcinoma.

New modalities such as Intraductal US (IDUS) and spiral CT have been used for tumour evaluation. IDUS was significantly superior to EUS and CT scan. (100% vs 59.3% vs 29.6% respectively). Sensitivity and specificity rates for IDUS and EUS were 100% versus 62.5% and 75% versus 50%, respectively. Overall accuracy rate in tumour diagnosis for IDUS was reported significantly superior to EUS (89% vs 56%) \[[@B35]\]. However in our hands as well as in other studies \[[@B25],[@B27]-[@B29],[@B31],[@B33]\] overall diagnostic accuracy for tumour detection was higher reaching levels as high as 84%. The reason for the relatively low accuracy of EUS in this study compared to the rest is not evident. IDUS appears to be the most effective imaging method in visualizing and staging tumors of the papilla but this remains to be confirmed by other studies. Combining ERCP with a catheter probe sonography offers a new diagnostic modality that has some potential advantages for local staging of small tumours of the papilla \[[@B35]-[@B37]\].

The sensitivity and specificity of another diagnostic modality, spiral CT, for detecting involvement by the tumour of the superior mesenteric vein, portal vein and lymph nodes have been reported similar to that of EUS \[[@B26]\]. However this was not true in our hands. We think that this discrepancy was due to the small number of our patients undergoing spiral CT (7/20).

In conclusion compared with classical imaging procedures like US and CT scan, EUS is an effective method for the evaluation of the extent of invasion of ampullary carcinoma as well as for the involvement of regional lymph nodes before operation. Spiral CT scan may be combined with EUS in order to evaluate distal metastases.

Competing interests
===================

None declared

![Kaplan-Meier survival curve of operated patients](1471-2482-2-1-5){#F5}

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

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Background
==========

Microbial cholesterol oxidases (EC 1.1.3.6) (COX) catalyze the oxidation and isomerization of cholesterol to 4-cholesten-3-one. Interest in these enzymes mostly relies in their utility in the determination of cholesterol in biological samples such as serum and foods \[[@B1]\], and also in the bioconversion of a number of 3β-hydroxysteroids in organic solvents \[[@B2]\] and in reverse micelles \[[@B3]\] (for a recent review see \[[@B5]\]). Since earliest reports on crude preparations from *Mycobacterium sp.*\[[@B4]\], cholesterol oxidases have been described in a number of bacteria and fungi \[[@B5]\]. Enzymatic properties of cholesterol oxidase from *Rhodococcus* strains (some of which named formerly as *Nocardid*) are particularly suitable for use in the analytical determination of cholesterol, in which the hydrogen peroxide formed is used in a chromogenic reaction catalyzed by horseradish peroxidase \[[@B6]\].

The *Rhodococcus* enzyme has been usually reported to be membrane bound, extractable from whole cells by treatment with detergents or trypsin, although no phospholipids are detected in the enzyme extracts \[[@B7]\]. More recent reports have demonstrated the production of both extracellular and cell-bound cholesterol oxidase by strains of this genus such as *Rhodococcus* sp. GK1 \[[@B8]\], *R. erythropolis* ATCC 25544 \[[@B9]\] and the pathogenic specie *R. equi*\[[@B10],[@B11]\].

The kinetics of enzyme synthesis at both bench and large scale by *Nocardia rhodochrous* (renamed as *Rhodococcus rhodochrous*), a strain that produces only a cell-bound COX, has been studied and the growing conditions for bacterial enzyme synthesis in fermentor were defined \[[@B12]\].

Due to the high cell-bound to extracellular ratio of cholesterol oxidase produced by *Rhodococcus,* even in those strains that also produce extracellular enzyme, the use of detergents is compulsory in the cost-effective extraction of this enzyme. The properties of protein extraction and purification combine in polyoxyethylene type detergents whose cloud point is in the biocompatible range \[[@B13]\]. For instance, Triton X-114 is as effective as Triton X-100 to extract membrane proteins, but its cloud point in semidiluted solutions (temperature at which the detergent solubility decreases sharply and a liquid-liquid phase separation is produced) is 23°C as compared to 65°C of Triton X-100. Therefore, extracted proteins partition between a detergent-rich phase and a detergent-depleted phase thus occurring protein purification. Protein purification has been accomplished successfully from either animal cells and organelles, plant tissues and microbial cells \[[@B13]-[@B15]\]. Triton X-114 at 1%w/v in buffers has been utilized to study the partitioning behavior of commercial cholesterol oxidase from several bacterial sources, resulting in partitioning toward the detergent rich-phase in all cases \[[@B16]\]. The polyoxyethylene detergent C12EO5 added the to a non-clarified culture of *Nocardia rhodochrous* was used for direct solubilization and extraction of the cell-bound cholesterol oxidase followed by phase separation \[[@B17]\]. A four-fold preconcentration and five-fold purification were achieved in optimal conditions. Due to the high cost of C12E05 these authors tried the cheaper four narrow range ethylene oxide surfactant C12-C18E05 \[[@B18]\] which was found equally suitable for direct solubilization and extraction of cell-bound cholesterol oxidase, thus this system was expanded to pilot scale \[[@B19]\].

In a previous work \[[@B9]\] we described the cell-bound and extracellular cholesterol oxidase activities from *R. erythropolis* ATCC 25544, achieving in optimal conditions 55% cell-bound and 45% extracellular activity. Their enzymatic properties strongly supported the idea that the particulate and the extracellular cholesterol oxidases are two different forms of the same enzyme with an estimated molecular mass of 55 kDa. In this work we optimize the culture conditions in a 2-liter fermentor of this extracellular cholesterol oxidase producer strain and carry out the extraction, partial purification and concentration of both types of cholesterol oxidase by using Triton X-114 phase separation. The results obtained are very promising for the use of this strain and this technique in the industrial processing of bacteria to obtain cholesterol oxidase.

Results and discussion
======================

Batch cultivation of *R. erythropolis* (ATCC 25544)
---------------------------------------------------

The bacteria were grown on the GYS medium in a 2-liter scale fermentor in batch mode operation under pH and temperature controlled conditions. Under this conditions the cell yield was doubled (9.5 mg/ml vs. 4.8 mg/ml dry cell weight) and the cultivation time was reduced to one third (60 vs. 180 hours) as compared with shaken flasks. These results are in good agreement with the literature \[[@B12]\]. We found that addition of 2 g/l cholesterol to the culture broth \[[@B12]\], prepared as an aqueous emulsion with the aid of Tween 80 at a weight ratio 2:1 results in a high yield of COX production \[[@B9]\], but the preparation procedure of that emulsion had a marked influence in the final enzyme yield, although not on the cell weight, as seen in Table [1](#T1){ref-type="table"}. The spray-dry method resulted advantageous because the cholesterol :Tween 80 emulsion formed readily and COX production increased in overall by three times with respect to the preparation of the  80 mixture at the flame. Enzyme production improvement resulted larger as cell-linked (3.8-fold) than as extracellular (2.3-fold). This overall increase of COX production can be due to a better availability of cholesterol to the cell since particle size obtained by spray-dry is smaller.

::: {#T1 .table-wrap}
::: {.caption}
######

Effect of the cholesterol emuIsification method on the production of COX.
:::

                                          **COX activity (U/ml)^\*^**
  --------------------------------------- ----------------------------- ------------------- ------------------------
  **Emulsification cholesterol method**   **Cell-linked**               **extracellular**   **Dry weight (mg/ml)**

  Spray-dry                               230                           140                 8.75
  At the flame                            60                            60                  9.05
  Improvement                             3.8                           2.3                 0.97

^\*^Enzymatic activity figures correspond to 70 hours of fermentation.
:::

Results from a typical batch fermentation are shown in Figure [1](#F1){ref-type="fig"}. Three stages can be differentiated during the fermentation process, (i) A first stage (0--16 h), in which 02 consumption increases continuously and HCl is consumed to keep the pH constant to 6.75. Buckland et al. \[[@B12]\] found that pH of the culture rose by 0.5 units in the first part of growth, and then fell. An exponential increase of cell mass is observed and low levels of COX activity appear linked to cells, (ii) The second stage (16--45 h) is characterized by a strong 02 consumption and a consumption of base. In this phase aerobic metabolism drives the cell growth but the growth rate is certainly limited by the O~2~ availability -- note pO~2~ is nearly zero under continuous stirring and air supply-. This stage is also characterized by a high rate of COX production of both types, cell-linked and extracellular, (iii) The third stage (45 h to the end of the process) a second phase of consumption of acid was recorded whereas pO~2~ increased again to reach saturating levels. The greatest increase of cell-linked COX production was observed in this stage whilst extracellular COX production stopped.

![Characterization of the *R. erythropolis* fermentation process: biomass and production of cell-linked and extracellular COX. Enzyme activities are given as units/ml of cell culture. The data shown are from a single experiment but are representative of three separate replicates.](1472-6750-2-3-1){#F1}

The profile of fermentation was very similar to that obtained by Buckland et al. \[[@B12]\] but differed in the accumulation of extracellular COX: the strain of Nocardia (NCIB 10554) used by these authors produced very low levels of extracellular enzyme while the strain tested in this work produces high levels. They also tested the effect of dissolved oxygen tension on the production of COX and found that in limiting conditions of oxygen supply the production of cell-linked COX was low. As seen in Figure [1](#F1){ref-type="fig"}, when oxygen supply is limiting (in the second stage) the rate of cell-linked COX production decreases, however is in these conditions when extracellular COX production takes place. Thus, there seem to be some relation between dissolved oxygen tension and extracellular COX production by the strain used in this work.

The results obtained are coherent with those presented in a previous study in shaken flasks \[[@B9]\], where extracellular COX production is large and arises from the partial solubilization of the cell-linked enzyme \[[@B20],[@B21]\]. After 70 hours of fermentation the total enzyme activity obtained was ca. 360 U/ml, being 230 U/ml cell-linked and 130 U/ml extracellular, thus the cell-linked to extracellular ratio is 1.26. This ratio in shaken flasks ranged from 1.26, using the same amount of Tween 80 as in this work (0.1%), to 1.38, using 1% Tween 80, but in the latter the overall yields of COX production were 7-fold smaller \[[@B9]\]. The overall yield obtained in this work is comparable to that obtained by Buckland et al. \[[@B12]\] and by Minut et al. \[[@B17]\] but larger than that of Cheetham et al. \[[@B22]\]. Watanabe et al \[[@B24]\] compared the cell-linked and extracellular COX production of 31 strains of the genus *Rhodococcus* and *Nocardia* and found that among the best extracellular COX producers, the strains *Rhodococcus* sp. N° 31 and *R. equi* N° 24, displayed the highest cell-linked to extracellular ratio, 1.32 and 2.68 respectively.

Use of Triton X-114 for extraction and purification of COX
----------------------------------------------------------

In most of the available wild producer strains, COX behaves as a cell-linked enzyme, which is particularly true in the genus *Rhodococcus*\[[@B7],[@B8],[@B23]\]. Significant levels of extracellular COX have been described to be produced by the pathogenic species *R. equi*\[[@B10],[@B11],[@B24],[@B25]\] and also by *R. erythropolis*\[[@B9]\] and *Rhodococcus* sp. \[[@B8],[@B24]\] in certain culture conditions.

Several authors have investigated the ability of different detergents to disrupt lipid-protein associations and to release cell-linked COX in its native state. The use of Triton X-100 has been largely accepted \[[@B7],[@B9],[@B12],[@B26],[@B27]\] but other polyoxyethylene type non-ionic detergents whose cloud point is in the biocompatible range can be used for COX solubilization and purification \[[@B17]\].

Table [2](#T2){ref-type="table"} shows the amount and the percentage of cell-linked COX extracted by either Triton X-100 or Triton X-114 at several detergent concentrations. COX could be extracted from cells and solubilized by both detergents at 1% w/v. The % of recovery is of the same order as previously reported for these detergents \[[@B17]\]. The increase of the Triton X-114 concentration from 1 to 3% w/v resulted in concomitant increases of 1.5-fold the total activity, 2.6-fold the specific activity and a % of recovery above 90%. A Triton X-114 concentration as high as 6% w/v does not improve significantly the recovery nor the specific activity. Phase separation was further induced to gain COX purity in extracts and in the broth independently.

::: {#T2 .table-wrap}
::: {.caption}
######

Cell-linked COX extraction by Triton X-100 and Triton X-114 detergents.
:::

  **DETERGENT**     **COX activity (UE/ml)**   **% recovery**   **COX activity (UE/mg Prot)**
  ----------------- -------------------------- ---------------- -------------------------------
  TRITON X-1001%    234.2                      59.3             1306.9
  TRITON X-114 1%   243.4                      61.6             566.9
  TRITON X-114 2%   274.5                      69.5             860.5
  TRITON X-114 3%   363.8                      92.1             1466.9
  TRITON X-114 6%   365.0                      92.4             1674.3
:::

The cell-free extract of Triton X-114 was subjected to phase separation as such. The culture broth was first supplemented with Triton X-114, well dissolved at 4°C and then warmed up to induce detergent phase separation. Figures [2a](#F2){ref-type="fig"} and [3a](#F3){ref-type="fig"} show the distribution of enzyme activity in each phase (detergent-depleted and -rich) for each of the COX sources (cells extract and culture broth) respectively.

![Distribution of COX activity among detergent depleted and detergent rich phases after induction of phase separation of culture broth supplemented with the indicated concentration of Triton X-114. (a) Total activity; (b) Specific activity.](1472-6750-2-3-2){#F2}

![Distribution of COX activity among detergent depleted and detergent rich phases after induction of phase separation of cell extracts done with the indicated concentration of Triton X-114. (a) Total activity; (b) Specific activity.](1472-6750-2-3-3){#F3}

As Triton X-114 concentration is increased, COX partitions towards the detergent rich phase, increasing its specific activity (Figures [2b](#F2){ref-type="fig"} and [3b](#F3){ref-type="fig"}) thus resulting in enzyme purification and also in enzyme concentration since the volume of the detergent-rich phase is much lower than the initial volume. The 1% concentration of detergent was an exception to this rule since COX partitioned toward the depleted phase under our working conditions. Partitioning of commercial COX in buffers containing 1% Triton X-114 occurred toward the rich phase and was very influenced by the buffer concentration \[[@B16]\]. Therefore, it seems that the composition of phase separation media is extremely important to the partitioning of particular proteins.

The purification was made evident by running samples of COX from cells and culture broth in SDS-PAGE gels. Figure [4](#F4){ref-type="fig"} shows that in both cases the detergent-rich phase was enriched in some proteins, including COX, whereas the depleted phase showed other different protein bands.

![SDS-PAGE of COX fractions using 3% Triton X-114 for extraction, purification and concentration, (a) Cell extracts: lane 1, Mw markers; lane 2, commercial COX; lane 3, total extracted proteins; lane 4, proteins in detergent depleted phase; lane 5, proteins in detergent rich phase, (b) Culture broth: lane 1, Mw markers; lane 2, commercial COX; lane 3, proteins in detergent rich phase; lane 4, total proteins in culture broth; lane 5, proteins in detergent depleted phase. Arrows indicated the COX band.](1472-6750-2-3-4){#F4}

An exceptional result was obtained when performing COX purification from the culture broth supplemented with a 6% w/v Triton X-114. The total activity recovered after phase separation was ca. 3.5-fold that measured in the broth before phase separation. This result suggests that soluble COX produced by the culture is not fully active and that it can be activated by a treatment with 6% Triton X-114 but not with 4% or less. Further increase of Triton X-114 concentration results in no improvement with respect to 6% (results not shown). This phenomenon was not observed with COX extracted from cells, therefore the enzyme most likely exists in a fully active form in the cells.

We have shown previously that cell-linked and soluble COX from the same strain are almost indistinguishable as judged by some enzymatic properties such as kinetic parameters, electrophoretic mobility of the native active enzyme and thermostability \[[@B9]\]. Now we show evidence of a differential characteristic of soluble COX as compared to cell-linked: the activation by 6% Triton X-114.

The observed phenomenon accepts in principle several explanations: (i) all the soluble COX molecules become activated by 3.5-fold due to a detergent effect on the protein conformation, (ii) a fraction of soluble COX is active and a fraction 3.5-fold larger is fully inactive, but can be activated due to a detergent effect on the protein conformation, (iii) an inhibitor is removed as a consequence of detergent treatment. From the first hypothesis it could be expected some difference between both enzyme forms at least at kinetic level, which we did not observe in previous studies, although it cannot be discarded. The inhibitor hypothesis is perhaps less likely since the activation effect might have been observed at all concentrations of Triton X-114 and gradually. The second hypothesis may be the most likely according to our previous results since we characterize only active enzyme and not the enzyme protein. In that case it may be hypothesized that there is an active form of COX able to both interact with components of the cell membrane or the cell wall to remain cell-linked, and to stay soluble in the culture broth, and there is an inactive form which is soluble in the culture broth. Reversion of inactive to active is induced by a high detergent concentration, which may provide an environment resembling that of cell membranes or cell walls. The active soluble form secreted by bacterial cells might eventually and reversibly turn into inactive soluble COX. When detergent concentration of the 6% rich phase was lowered by dilution the specific activity did not change, therefore the conversion of active to inactive must be very slow, that is, the existing enzyme forms are not in equilibrium. In any case, the characterization of this activation phenomenon requires further studies that are now under progress in our laboratory.

The enzymatic properties exhibited by COX have been shown to depend on the method of extraction from cells, either with Triton X-100, with buffer or with trypsin \[[@B7]\]. The extracted enzyme could be interconverted from one to another with appropriated treatments such as addition or removal of 0.5% Triton X-100. So they showed evidence for the existence of different isoforms of COX from *Nocardia rhodochrous* (renamed as *Rhodococcus rhodochrous*). These authors also pointed out that no phospholipids were co-extracted even when using Triton X-100 and suggested that cell-linked enzyme is anchored through a hydrophobic tail that interacts with naturally occurring surfactants of the cell wall.

Taking that work into account and our own results it may be proposed that COX shed from cell walls aided by bacterial surfactant solubilization becomes the extracellular enzyme and so its level might be related with the production of bacterial surfactants.

The effect of Triton X-114 phase separation step on the purification and concentration of COX is summarized in Table [3](#T3){ref-type="table"}. Taking together purification, concentration and % of recovery, the best results were obtained when using Triton X-114 at 3% w/v for the cell-linked and at 6% for the extracellular COX. Minuth et al. \[[@B17],[@B18]\] achieved 5-fold purification and 4-fold concentration adding the detergent pentaethyleneglycol mono *n*-dodecyl ether (C12EO5) to an non-clarified culture of *Nocardia rhodochrous.* Our results, 11.6-fold purification and 20.3-fold concentration indicate that Triton X-114 should be highly recommended for use with a clarified culture broth of an extracellular COX producer strain.

::: {#T3 .table-wrap}
::: {.caption}
######

Purification and concentration of COX during Triton X-114 phase separation.
:::

                             **Cells extract of 3%Triton X-114 w/v**   **Culture broth with 6% Triton X-114 w/v**
  -------------------------- ----------------------------------------- --------------------------------------------
  **Purification fold**      1.67                                      11.6(2.52)^b^
  **Concentration factor**   2.56                                      20.3 (4.38)^b^
  **% recovery**             76 (70)^a^                                312 (65)^b^

^a^Recovery in this step and, in parenthesis, with respect to cells. ^b^ Figures in parenthesis correspond to purification, concentration and recovery in this step as if no activation occurred, calculated on the basis of remaining activity in the upper detergent-depleted phase
:::

The quantification of COX partitioning in the Triton X-114 two-phase system was accomplished by determining its partition coefficient on the basis of enzyme activity. To that, concentration of COX activity in each phase has been determined by measuring enzyme activity and phase volumes after phase separation. The partition coefficient is defined as activity concentration in the rich phase over activity concentration in the depleted phase:

K= \[COX\]~rich~ / \[COX\]~depleted~

Results are shown in Figure [5](#F5){ref-type="fig"}. Concerning the enzyme extracted from cells, partition coefficient reaches an optimum at 3% w/v detergent, decreasing at higher detergent concentrations. We do not have direct evidence to explain this decrease but a reason for it could be a change in the composition of the rich phase that may contain more components of the bacterial cell wall extracted at high detergent concentration, thus interfering in the partitioning of COX. In the case of the extracellular COX the higher the detergent concentration the larger the partition coefficient. As seen in table [4](#T4){ref-type="table"}, the rich phase is quite more compact after phase separation in the culture broth than in the cell-extract and its relative volume is not proportional to the detergent concentration. Thus the rich phase is less hydrated, that is, more hydrophobic, with the culture broth than with the cell-extract and the more hydrophobic the higher the detergent concentration. As a result the partition coefficient for COX in the culture broth increases with the detergent concentration. Phase separation of Triton X-114 is affected by the presence of other surfactants such as Triton X-45 \[[@B28]\] and polyols such as glycerol \[[@B29]\], thus bacterial surfactants extracted during detergent treatment may affect phase separation as well. Natural surfactants may be also present in the broth but at a much lower concentration, therefore having less influence on the phase separation.

![Partition coefficient of cell-linked (•) and extracellular (•) COX in a Triton X-114 phase separation system.](1472-6750-2-3-5){#F5}

::: {#T4 .table-wrap}
::: {.caption}
######

Partitioning of cell-linked and extracellular COX after phase-separation of Triton X-114
:::

                                                **COX total units**   **COX activity (U/ml)**   **K**
  ------------------- ------------------------- --------------------- ------------------------- ------------ ---------------- -------
  \% Triton X-114     \% volume of rich phase   Rich phase            Depleted phase            Rich phase   Depleted phase

  **Cell-linked**

  1                   10.8                      8.5                   234.8                     78.7         263.2            0.30
  2                   15.9                      137.9                 136.5                     867.3        162.3            5.34
  3                   29.6                      276.5                 87.1                      934.1        123.7            7.55
  4                   37.5                      278.0                 86.5                      741.3        138.4            5.35
  6                   41.8                      280.0                 85.0                      669.8        146.0            4.58

  **Extracellular**

  1                   6.4                       5.0                   113.7                     78.2         121.5            0.64
  2                   9.3                       22.8                  99.7                      245.7        109.7            2.24
  3                   10.7                      58.8                  69.4                      550.5        77.7             7.08
  4                   12.8                      70.5                  55.1                      552.3        63.1             8.75
  6^a^                15.3                      84.0                  41                        547.9        48.4             11.32
  6                   15.3                      390                   41                        2544         48.4             52.60

^a^COX units in rich phase calculated as if no activation occurred: total units before partitioning (125) -- units in depleted phase (41)
:::

The partitioning of commercial COX from *Nocardia erythropolis* and *Pseudomonas sp.* has been shown to depend on the detergent partitioning, since factors that affected the Triton X-114 distribution, such as temperature of partitioning, pH and phosphate concentration of the buffers, affected in the same way the distribution of enzyme \[[@B16]\]. Thus our results are in agreement with those findings. The reason for the different behavior of the detergent packing in cell extracts and in broth may lie in the fact that detergent partitioning is affected by physicochemical factors such as the presence of polyols, lipids, surfactants, etc \[[@B13]\], which can be present in cell extracts but not in the cell culture.

Conclusions
===========

From a technological point of view it is simpler to handle an extracellular enzyme than a cell-linked one. The amount of extracellular COX of the non-pathogenic strain *R. erythropolis* ATCC 25544 obtained in a 1.5 1 batch fermentation represents a 36% of the activity (130 out of 360 U/ml) when measured directly from the broth, but after the 6% Triton X-114 treatment and phase separation it represents a 65% of the total activity (442 out of 672 U/ml). In addition, active COX becomes 11.6 times purer and 20.3 times more concentrated. These results may make attractive and cost-effective the use of this bacterial strain and the Triton X-114 phase separation in a 6% w/v for the industrial production of COX used in serum and food cholesterol analysis. The purification step based on Triton X-114 phase separation should be followed by further steps, such as ion-exchange chromatography, which can combine non-ionic detergent removal and protein purification in one step, in order to obtain a preparation of COX suitable for analytical applications \[[@B16]\].

Investigations to improve the percentage of extracellular COX are currently under progress.

Materials and methods
=====================

Commercial cholesterol oxidase from *R. erythropolis* was purchased from Boehringer Mannheim (25 U/mg). Cholesterol from lanolin, cholesterol from human gallstones, Triton X-100 and Triton X-114 were purchased from Fluka. Triton X-114 was condensed three times in 5 mM sodium phosphate buffer pH 7.5 \[[@B30],[@B31]\]. The detergent phase of the third condensation had a concentration of 25% w/v TX-114 and was used as the detergent stock solution for all the experiments. The Triton X-114 concentration was determined from its absorption at 278 nm (A~278~ = 1.25 for 0.05% w/v) \[[@B29]\]

Microorganism and culture conditions
------------------------------------

The strain used in this work was *R. erythropolis* ATCC 25544 which was routinely maintained in the laboratory by periodic subculturing in GMP medium \[[@B20]\] consisting of 0.1 g/l glucose, 0.02 g/l yeast extract, 0.04 g/l peptone, 0.04 g/l meat extract, 0.05 g/l NaCl, 0.0025 g/l MgSO4 and 0.25 g/l agar.

The microbial production of cholesterol oxidase was assessed as previously described by us \[[@B9]\].

Cells were grown in GYS medium in a 2 1 reactor (BIOSTAT B from B. Braun Biotech Ltd.) with a working volume of 1.5 1. Air was supplied at 2.6 vol/vol/min; pH was set constant to 6.75 and temperature to 29°C. The GYS medium is a modification of the mineral medium described by Buckland \[[@B26]\] that consisted of 10 g/l glycerol, 20 g/l yeast extract, 2 g/l (NH~4~)~2~SO~4~, 2 g/l K~2~HPO~4~, 0.01 g/l CaCl~2~.2H~2~O, 0.01 g/l FeSO~4~.7H~2~O, 0.1 g/l MgSO~4~.7H~2~O. When this culture reached a dry weight of ca. 1.0 mg/ml, an aqueous suspension of Tween 80/cholesterol was added to a final concentration of 0.2% cholesterol and 0.1% Tween 80.

An aqueous suspension of cholesterol was prepared in two ways; at the flame and by a spray-dry method. (i) In the first method, cholesterol and Tween 80 were mixed by heating at the flame until total dissolution of solids, then water was added to form an emulsion by vigorous shaking for 1 hour. (ii) In the second, cholesterol and Tween 80 were co-dissolved in diethyl ether; the solvent was then removed by spray drying and the solid material was recovered and used to readily prepare an stable aqueous suspension.

Extraction and partial purification of cholesterol oxidase
----------------------------------------------------------

The extraction of cell-linked COX by using Triton X-100 was as described previously \[[@B9]\]. For the cell linked COX extraction and purification by the Triton X-114 method, the extract obtained in cold after removal of cells by centrifugation was submitted to temperature-induced phase separation. The coalescence of the detergent was facilitated by warming up to 37°C for 15 min that was followed by the sharp separation of the two resulting phases by spinning at 4000 g for 15 min at 25°C. Both phases, the lower detergent-rich and the upper detergent-depleted were assayed for both enzyme activity (see below) and protein \[[@B32]\]. Using Triton X-114 also purified the extracellular COX. The cold culture broth was supplemented with Triton X-114 to the desired final concentration and detergent was completely dissolved at 4°C. Phase separation was induced as above.

Enzyme assay
------------

Cholesterol oxidase activity was assayed by a modification of the method of Allain et al. \[[@B1]\] as described previously \[[@B9]\]. One unit of activity was defined as the amount of enzyme that converts 1 μmole of cholesterol/min at 37°C. All samples were diluted before enzyme assay to a final Triton X-114 concentration of 0.1% to avoid detergent interference with the assay \[[@B16]\].

SDS-PAGE
--------

The protein extracts were prepared as described previously \[[@B9]\]. SDS-PAGE electrophoresis \[[@B33]\] was carried out at 200 volts at 25° in a Mini Protean cell (Bio-Rad, Richmond, California). The gels were developed by using the silver staining technique.

Acknowledgements
================

M.S. is a predoctoral fellow from the Spanish Ministry of Education (plan F.P.I.). This work was supported by grants BIO394-0451 and AGF99-0396 from C.I.C.Y.T. and PIB-95/03 from Comunidad Autónoma de Murcia.


Background
==========

During the last 30 years, the incidence of allergic diseases, including pollen allergy and allergic asthma, has increased in industrialized countries \[[@B1]-[@B7]\]. Epidemiological studies suggest that the increase in the prevalence of allergic diseases may be associated with environmental exposures, lifestyle, temporal development of the immune system, and genetics \[[@B8]\]. Diesel exhaust, which is one of the major sources of environmental pollution in the 20th century, has been suspected to accelerate pollen allergy and allergic respiratory diseases \[[@B9]-[@B12]\], because diesel exhaust particles (DEP) may act as an adjuvant \[[@B13]-[@B22]\] or directly induce activated mast cells to degranulate \[[@B23]\].

Previous studies addressed the possibility that inhalation of diesel exhaust during pregnancy disrupted differentiation of the thymus in the fetus due to the accumulation of testosterone \[[@B24],[@B25]\]. Testosterone-binding cells have been found in the thymus of 18-day-old fetuses and testosterone can influence the function of specific thymic epithelial cells \[[@B26]-[@B28]\]. Such immunodeficiency of the thymus, especially of suppressor T cells, elevates serum immunoglobulin E \[[@B29]-[@B32]\]. These findings strongly suggest that the inhalation of diesel exhaust during the differentiation periods suppresses proliferation of the thymus cells and contributes to the development of allergies in postnatal life.

This study was conducted to pursue three objectives. First, we sought to determine the impact that diesel exhaust inhalation during the differentiation periods of the immune system had upon the elevation of immunoglobulin E against Japanese cedar pollen. To show this, time of exposure to diesel exhaust was based on the developmental process of the thymus \[[@B33],[@B34]\]. The second objective was to determine which substances -- particulate matter or the gaseous phase present in diesel exhaust -- are responsible for the elevation of IgE. The third objective was to determine the mechanism by which the inhalation exerts its toxicity by measuring the serum levels of hormones.

Materials and methods
=====================

Generation of diesel exhaust
----------------------------

Diesel engine exhaust was generated by running a 309 cc engine (Model NFAD50, Yanmar Diesel Co., Osaka, Japan) at 2,400 rpm. Exhaust was diluted with clean air in a dilution tunnel and then drawn into the inhalation chamber (particulate matter = 1.73 mg/m^3^, nitrogen dioxide = 0.79 ppm). For the filtered group, most of the diesel soot particles in whole exhaust were removed by HEPA filtration (ATM 3QA, Nippon Muki Co., Tokyo, Japan). After filtration, 99.9998% of particles measuring 0.05 μm or more were eliminated. Ventilation was maintained by 15 air exchanges per hour. Concentrations of nitrogen dioxide and nitrogen monoxide were continuously monitored with a chemiluminescent analyzer (Model 8440, Monitor Labs Co., San Diego, CA). Gravimetric measurements of the particulate matter were conducted daily using an automatic beta-ray dust-mass monitor (Model BAM-102, Shibata Scientific Technology Co., Tokyo, Japan). Measurement of particle sizes with a particle fractionating sampler (Andersen Type low pressure impactor LP-20, Tokyo Dylec Co., Tokyo, Japan) confirmed that more than 90% of the particulate matter in the diesel exhaust measured less than 0.05 μm.

Experiment 1
------------

### Animals and treatments

Forty-three pregnant Fischer rats (F344/DuCrj) obtained from Charles River Japan (Kanagawa, Japan) were divided into seven groups on the sixth days of pregnancy. The experiment was conducted using only male pups from those mothers. Group 1 was exposed to clean air during all of the experimental period (*Control*). Group 2 was exposed to total diesel exhaust from the 7th day of gestation (day of impregnation = day 0) until 3 days after birth, then exposed to clean air until days 49, 82 and 96 after birth. (*Total-C-C*). Group 3 was exposed to filtered exhaust from the 7th day of gestation until 3 days after birth, then exposed to clean air until days 49, 82 and 96 after birth (*Filtered-C-C*). Group 4 was exposed to diesel exhaust from the 4th day after birth through the 22nd day after birth. The group was exposed to clean air during the other periods of the experiment (*C-Total-C*). Group 5 was exposed to filtered exhaust from the 4th day after birth through the 22nd day after birth. The group was exposed to clean air during the other periods of the experiment (*C-Filtered-C*). Group 6 was exposed to diesel exhaust from day 23 after birth until day 41. The group was exposed to clean air during the other periods of the experiment (*C-C-Total*). Group 7 was exposed to filtered exhaust from day 23 after birth until day 41. The group was exposed to clean air during the other periods of the experiment (*C-C-Filtered*). Each group had the same exposure period, 19 days, to diesel exhaust. The number of pregnant rats for groups 1, 2, 3, 4, 5, 6 and 7 were 7, 7, 7, 6, 6, 5 and 5, respectively. Litter sizes ranged from 6 to 11. The total number of pups (male: female) of each group from the mothers were 53 (28:25), 50 (24:26), 54 (25:29), 47 (22:25), 44 (23:21), 43 (22:21) and 45 (20:25), respectively. The experimental protocol is summarized in Figure [1](#F1){ref-type="fig"}.

![Experimental protocol. *Control,* exposed to clean air; *Total-C-C,* exposed to total diesel exhaust from the 7th day of gestation until 3 days after birth; *Filtered-C-C,* exposed to filtered exhaust from the 7th days of gestation until 3 days after birth; *C-Total-C,* exposed to diesel exhaust from the 4th day after birth through the 22nd day after birth; *C-Filtered-C,* exposed to filtered exhaust from the 4th day after birth through the 22nd day after birth; *C-C-Total,* exposed to diesel exhaust from day 23 after birth to day 41; *C-C-Filtered,* exposed to filtered exhaust from day 23 after birth to day 41.](1471-2393-2-2-1){#F1}

Each group of animals was maintained in an inhalation chamber (1.6 m^3^) at 24 ± 2°C and 55 ± 5% humidity on a 12-hr light: 12-hr dark illumination schedule. The diet was standard rat chow containing 1.03% calcium, 0.70% phosphorus and 200 IU vitamin D3/100 g (MF, Oriental Yeast Co. Ltd, Tokyo, Japan). The pups were kept with their mothers until weaned at 22 days of age. At the same time, the young rats were divided into groups of males and females?\" the male rats were randomly assigned to groups of 6 to 8 housed in a single cage. All the animals were allowed free access to food and water. Exposure to diesel exhaust was 6 h daily from 10 a.m. to 4 p.m., for 19 days.

Body weights, thymus and spleen weights were measured and blood samples were collected to measure the titer of immunoglogulin from the abdominal aorta under ether anesthesia on day 49 before immunization and five days after the 3rd or 4th immunization.

### Allergens and immunization procedure

The crude Japanese cedar pollen allergens were kindly provided by Dr. Maejima through the Japan Automobile Research Institute (Tsukuba, Japan). The amount of protein in the crude Japanese cedar pollen allergens was 65 μg containing 17.4 μg of Cry j 1 and 1.73 μg of Cry j 2 per milliliter \[[@B35]\]. Intraperitoneal injection of 1 ml of 0.125 M ammonium bicarbonate containing 5 mg crude Japanese cedar pollen allergens mixed with 4 mg aluminum hydroxide gel was performed three or four times at 2-week intervals from the 49th day after birth.

### Measurement of anti-Japanese cedar pollen IgE antibody titers

The titers of IgE antibodies were measured by the Prausnitz-Kustner reaction (P-K reaction) in 12-week-old male Fischer rats (F344/DuCrj) obtained from Charles River Japan (Kanagawa, Japan). One tenth milliliter of serial two-fold dilutions of the test sera was injected intracutaneously for the passive sensitization. Forty-eight hours later, the rats were challenged intracutaneously with 0.5 μg of Cry j 1 (Hayashibara Biochemical Lab. Inc. Japan) and 0.25 μg of Cry j 2 (Hayashibara Biochemical Lab. Inc. Japan) in 0.02 ml of PBS. At the same time, 1 ml of 0.5% Evans blue was injected intravenously. The test was carried out in duplicate, and the P-K titer was expressed as the geometric means of the reciprocal of the maximal dilutions that elicited a reaction of 5 mm in diameter.

Enzyme-linked immunosorbent assay (ELISA) of anti-Japanese Cedar Pollen IgE antibodies was carried out by a modification of the method of Imaoka et al. \[[@B36]\]. Briefly, the wells of microtiter plates (Immulon 2, Dynatech) were coated with 100 μg/ml of a commercial anti-rat IgE mouse monoclonal antibody (Zymed, USA) diluted to 20 μg/mL with 0.1 M NaHCO~3~ (pH8.2) at 37°C for 3 h, and the plates were washed 5 times with phosphate buffered saline (PBS). The wells were then successively incubated at room temperature with the following reagents, washing 5 times between each pair of steps: 250 μL of Block Ace (Dainihon Seiyaku, Osaka, Japan) for blocking at 4°C overnight; 100 μL of serial 10-fold dilutions of the test and normal sera?\" 100 μL of biotinylated cedar pollen allergens (Biotin Labeled Cry j 1 and 2, Hayashibara Biochemical Lab. Inc. Japan) (1 μg/mL); 100 μL of streptavidin-peroxidase (Zymed, USA) diluted 1:1,000; 100 μL of substrate solution containing 3, 3\', 5, 5\'-tetramethylbenzidine (Morinaga, Japan). Absorbance was read at 450 nm with a microplate reader. IgE titers were expressed as the absorbance obtained by subtracting the absorbance of the serum from control group.

Experiment 2
------------

Eighteen pregnant Fischer rats were divided into three groups: control, total exposed and filtered exposed. The exposure period was from day 7 of pregnancy until 3 days after delivery, corresponding to *Control, Total-C-C* and *Filtered-C-C* in experiment 1. On day 4 after birth, blood samples of newborn rats were collected. The serum pooled from the male pups of the same group was used as a sample. Serum testosterone and estradiol levels were determined using Enzyme Immunoassay Kits (Cayman Chemical, USA). Exposure conditions were the same as aforementioned.

Suckling rats with their mothers were divided into three groups on day 4 after birth: control, total exposed and filtered exposed, corresponding to *Control, C-Total-C* and *C-Filtered-C* in experiment 1. The number of mother rats was six. On day 23 after birth, blood samples of weaned males were collected and hormonal levels were assayed.

Statistical analysis
--------------------

All reported values are expressed as means ± standard deviations (SD) or means ± standard errors (SE) except for testosterone and estradiol of 4-day-old rats in experiment 2. The differences between the groups were analyzed using the Dunnett\'s test and Steel\'s test. The rank-order correlation between IgE titers was measured by ELISA and those of the P-K reaction were analyzed by Spearman\'s method. P-values \< 0.05 were considered significant.

The treatment and care of the rats were carried out according to the protocol approved by the Animal Care and Use Committee of the Tokyo Metropolitan Research Laboratory of Public Health in a facility approved by the Japan Association for Accreditation of Laboratory Animal Care.

Results
=======

Experiment 1
------------

The average body weight, spleen and thymus weight of male rats from 7 groups, *Control, Total-C-C, Filtered-C-C, C-Total-C, C-Filtered-C, C-C-Total and C-C-Filtered,* on days 49, 82 and 96 are shown in Table [1](#T1){ref-type="table"}. The average weight of the thymus of non-immunized rats on day 49 was significantly lower in *Total-C-C, Filtered-C-C* and *C-Filtered-C* than in the control group (p \< 0.01, p \< 0.01, p \< 0.05, respectively). There were no differences in thymus weight between the total-exhaust-exposed group and filtered-exhaust-exposed group. The average weight of the thymus of rats on day 82 after the third immunization was significantly lower in *Total-C-C* and *Filtered-C-C* than in the control group (p \< 0.01, p \< 0.05, respectively). The average weight of the thymus of rats on day 96 after the fourth immunization was significantly lower in *Filtered-C-C* and *C-Filtered-C* than in the control group (p \< 0.05 for each). There were no significant differences in thymus weight between the total-exhaust-exposed group and filtered-exhaust-exposed group.

::: {#T1 .table-wrap}
::: {.caption}
######

Average body, spleen and thymus weight of male rats from each group on days 49, 82 and 96
:::

                 non-immunized: rats on day 49   82-day-old rats immunized three times   96-day-old rats immunized four times
  -------------- ------------------------------- --------------------------------------- -------------------------------------- ------------------- --- -------------- -------------- ---------------------- --- --------------- --------------- -------------------
  Control        8                               143.5 ± 10.5                            488.0 ± 20.5                           323.1 ± 25.5        8   238.7 ± 5.7    601.8 ± 28.3   235.0 ± 19.8           8   252.9 ± 13.4    625.0 ± 42.0    204.8 ± 18.6
  Total-C-C      8                               143.5 ± 11.3                            488.0 ± 38 .3                          276.5 ± 18.9 ^\*^   8   230.1 ± 9.3    601.0 ± 30.6   199.9 ± 25 .0 ^\*\*^   8   257.6 ± 17. 8   631.8 ± 41.7    186.5 ± 20. 3
  Filtcrcd-C-C   8                               132.7 ± 11.9                            464.1 ± 61.5                           272.9 ± 49.3 ^\*^   8   230.9 ± 11.5   588.2 ± 25.0   213.5 ± 26.7           8   249.9 ± 12.8    658.5 ± 30.4    179.6 ± 21.9
  C-Total-C      8                               136.1 ± 7.9                             462.0 ± 30.9                           308.2 ± 26.1        8   230.6 ± 8.5    575.1 ± 49.4   214.7 ± 36.6           6   256.8 ± 14.6    619.2 ± 44.3    210.5 ± 40.8
  C-Filtcrcd-C   7                               137.8 ± 7.1                             475.0 ± 25.8                           289.0 ± 23.5        7   232.5 ± 6.5    606.1 ± 32.5   214.5 ± 25.1           8   241.0 ± 17.0    608.5 ± 44.5    176.1 ± 13.7 ^\*^
  C-C-Total      8                               146.9 ± 6.4                             488.7 ± 25.7                           310.1 ± 17.8        7   239.7 ± 10.5   621.9 ± 14.7   243.1 ± 23.3           7   261.6 ± 11.5    648.8 ± 39.1    218.4 ± 24 .2
  C-C-Filtcrcd   8                               142.7 ± 11.7                            490.2 ± 32.9                           309.3 ± 19. 9       6   240.2 ± 9.5    620.0 ± 21.2   248.2 ± 19.5           6   260.3 ± 10.1    663.1 ± 69. 6   220.6 ± 16.0

Values are expressed as mean ± SD. ^\*^Different from *Control*, p \< 0.05; ^\*\*^ different from Control, p \< 0.01
:::

The individual anti-Japanese cedar pollen IgE antibody titer measured by the Prausnitz-Kustner reaction (P-K reaction) and the mean IgE titers in *Control, Total-C-C,* Filtered-C-C. C-Total-C, C-Filtered-C, C-C-Total and C-C-Filtered *after the third and fourth* immunization are shown in Figure [2](#F2){ref-type="fig"}. The geometrical means of IgE titers in *Control,* Total-C-C, Filtered-C-C, C-Total-C, C-Filtered-C, C-C-Total and C-C-Filtered were 32.0 ± 6.9, 181.0 ± 1.5, 166.0 ± 1.4, 49.4 ± 4.0, 43.1 ± 6.5, 4.0 ± 1.0 and 6.3 ± 2.0 after the third immunization, and 64.0 ± 2.7, 469.5 ± 1.6, 332.0 ± 1.7, 380.4 ± 1.7, 394.8 ± 1.7, 115.9 ± 1.3 and 57.0 ± 2.8 after the fourth immunization, respectively. Log~2~ P-K IgE titers of each group are shown in Table [3](#T3){ref-type="table"}. There were significant differences between *Total-C-C, Filtered-C-C, C-Total-C, C-Filtered-C* and *Control* after the fourth immunization (p \< 0.01, p \< 0.01, p \< 0.05, p \< 0.01, respectively). There were no differences between the total-exposure and filtered-exposure conditions.

![Anti-Japanese cedar pollen IgE titers measured by P-K reaction. • IgE titers in the individual rat; ^\*^ geometrical means of each group](1471-2393-2-2-2){#F2}

::: {#T2 .table-wrap}
::: {.caption}
######

Organ weight and serum hormone levels of 4- and 23-day-old male rats from each group.
:::

                    No.   body weight (g)   spleen (mg)         thymus (mg)         testosterone (pg/ml)   estrogen (pg/ml)
  ----------------- ----- ----------------- ------------------- ------------------- ---------------------- ------------------
  4-day-old rats
   *Control*        20    10.14 ± 1.11      51.4 ± O.1          24.1 ± 1.1          166.6                  17.8
   *Total-C-C*      24    9.90 ± 0.68       46.3 ± 1.0 ^\*^     20.4 ± 1.4 ^\*^     390.1                  28.9
   *Filtered-C-C*   25    9.55 ± 0.86       42.1 ± 4.3 ^\*\*^   19.0 ± 1.5 ^\*\*^   255.8                  16.0
  23-day-old rats
   *Control*        10    32.35 ± 2.33      134.1 ± 16.1        108.4 ± 17.4        361.1 ± 139.4          39.8 ± 19.8
   *C-Total-C*      11    34.25 ± 3.61      144.5 ± 15.2        105.5 ± 15.1        497.4 ± 220.6 ^\*^     40.4 ± 26.8
   *C-Filtered-C*   8     32.84 ± 5.12      127.1 ± 20.1        93.2 ± 17.3 ^\*^    659.5 ± 356.2 ^\*\*^   52.5 ± 22.4

Values are expressed as mean ± SD. Testosterone and estrogen levels on day 4 were pooled serum from males. ^\*^ Different from Control, p \< 0.05; ^\*\*^ different from Control, p \< 0.01
:::

The average IgE titers measured by ELISA after the fourth immunization and expressed as optical density in *Control, Total-C-C, Filtered-C-C, C-Total-C, C-Filtered-C, C-C-Total and C-C-Filtered* were 0.47 ± 0.06, 0.79 ± 0.35, 0.86 ± 0.46, 0.80 ± 0.22, 0.56 ± 0.08, 0.46 ± 0.04 and 0.45 ± 0.03, respectively. The IgE titers in *Filtered-C-C* and *C-Total-C* were significantly higher than in *Control* (p \< 0.05, P \< 0.05, respectively). There was a significant rank-order correlation between the IgE titers measured by ELISA and those of the P-K reaction (p \< 0.01).

Experiment 2
------------

Table [2](#T2){ref-type="table"} shows the average body weight, spleen and thymus weight, as well as the serum levels of testosterone and estrogen of 4-day-old males exposed to clean air or diesel exhaust from the 7th day of gestation to 3 days after birth. The average spleen and thymus weights were significantly lower in *Total-C-C* (p \< 0.05, p \< 0.05) and *Filtered-C-C* (p \< 0.01, P \< 0.01) than in *Control.* The testosterone levels were higher in *Total-C-C* and *Filtered-C-C* than in *Control.* Table [2](#T2){ref-type="table"} also shows the average body weight, spleen and thymus weight and hormone levels of 23-day-old male rats exposed to clean air or diesel exhaust during the suckling period. The average thymus weight was significantly lower in *C-Filtered-C* than in *Control* (p \< 0.05). The testosterone levels were significantly higher in *C-Total-C* and *C-Filtered-C* than in *Control* (p \< 0.05, P \< 0.01, respectively). There were no significant differences between the total-exhaust-exposed group and filtered-exhaust-exposed group.

::: {#T3 .table-wrap}
::: {.caption}
######

IgE titers measured by P-K reaction against pollen in rats immunized three or four times
:::

                   Immunized three times   Immunized four times
  ---------------- ----------------------- ---------------------- --- -----------------
  *Control*        8                       5.0 ± 1.0              8   6.0 ± 0.5
  *Total-C-C*      8                       7.5 ± 0.2              9   8.9 ± 0.2^\*\*^
  *Filtered-C-C*   8                       7.4 ± 0.2              8   8.4 ± 0.3^\*\*^
  *C-Total-C*      8                       5.6 ± 0.7              6   8.6 ± 0.3^\*^
  *C-Filtered-C*   7                       5.4 ± 1.0              8   8.6 ± 0.3^\*\*^
  *C-C-Total*      7                       2.0 ± 0.0              7   6.9 ± 0.1
  *C-C-Filtered*   6                       2.7 ± 0.4              6   5.8 ± 0.6

Values are expressed as mean ± SD. Testosterone and estrogen levels on day 4 were pooled serum from males. ^\*^ Different from Control, p \< 0.05; ^\*\*^ different from Control, p \< 0.01
:::

Discussion
==========

The present study provides the first evidence that inhalation of diesel exhaust during the differentiation periods of the immune system accelerated the elevation of immunoglobulin E (IgE) against Japanese cedar pollen.

Elevation of IgE against Japanese pollen was detected in the rats exposed to total or filtered diesel exhaust from the 7th day of gestation until 3 days after birth (*Total-C-C, Filtered-C-C*). Previous studies addressed the possibility that inhalation of diesel exhaust during pregnancy disrupted the differentiation of the thymus in the fetus due to the accumulation of testosterone \[[@B24],[@B25]\]. In the present study, the testosterone levels, which were probably closely related to maternal hormone levels, were significantly higher in 4-day-old rats from *Total-C-C* and *Filtered-C-C* than the rats from *Control.* Testosterone-binding cells have been found in the thymus of 18-day-old fetuses. Testosterone can influence the function of specific thymic epithelial cells not only directly by acting on the thymus cells, but also indirectly by modulating the function of the thymus epithelial cells that bind testosterone \[[@B26]-[@B28],[@B37]\]. Other possibilities for the depletion of lymphoid cells are a reduction in the rate of bone marrow-derived prothymocyte entry into the thymus \[[@B12],[@B38]-[@B42]\], or a higher incidence of thymocyte apotosis \[[@B12],[@B43]-[@B45]\]. Since a certain number of lymphocytes in the spleen are from the thymus, decreased weight of the spleen on day 4 after birth in *T-C-C* and *F-C-C* might reflect disturbed or delayed development of the thymus. With regard to adjuvant effects of particles, \_particles deposited in the maternal organs might have acted as an adjuvant during the fetal and suckling periods and affected immune functions. There is a little possibility that diesel exhaust acted directly as an adjuvant for causing the elevation of IgE. The rats from the groups of *Total-C-C* and *Filtered-C-C* had been exposed to diesel exhaust through breathing for 3 days.

Elevated IgE was also detected in the rats exposed to diesel exhaust directly during the suckling period, *C-Total-C* and *C-Filtered-C.* Those rats had been exposed to diesel exhaust in two ways, through inhaled air and mother\'s milk. It has been indicated that inhalation of nitrogen dioxide, a strong oxidative gas contained in diesel exhaust, reduces T lymphocyte subpopulations \[[@B46]-[@B48]\]. The other possibility for hypersensitivity to pollen is the elevated steroid hormone level detected in 23-day-old rats from *C-Total-C* and *C-Filtered-C.* The influences from deposited particles in the maternal organs could not be neglected. The rats exposed to exhaust directly during weaning period, *C-C-Total and C-C-Filtered,* did not show an elevation of IgE, though those rats had a shorter interval between exposure and the first immunization compared to the rats from the other groups. Holladay et al. suggested \[[@B34]\] that developmental exposure to immunotoxicants may play a role in inducing or exacerbating hypersensitivity, which is long-lasting or permanent. It is reasonable to assume that inhalation could have influenced the differentiation period of the thymus, such as the initiation of hematopoiesis, migration of stem cells, the expansion of progenitor cells and the colonization of bone marrow and maturation to immunocompetence and induced hypersensitivity in later life.

The rats from *Total-C-C* and *Filtered-C-C* responded to a smaller dose of pollen than the rats from *C-Total-C* and *C-Filtered-C.* Judging from the decreased weight of the thymus and spleen on day 4 after birth, the impact of diesel exhaust inhalation on the development of the fetal immune system could be stronger than that on the neonatal immune system. Recently, it has been pointed out that the incidence of Japanese cedar pollinosis has increased not only in children but also in infants \[[@B49],[@B50]\]. There could be some connection between insufficient development of immune system during the fetal and neonatal periods because the mother has been exposed to polluted air and the increased prevalence of Japanese cedar pollinosis and atopic diseases in infants.

Regarding which substances were more responsible for insufficient development of the immune system and elevation of IgE, filtered-exhaust that included the gaseous phase and ultra fine particles measuring less than 0.05 μm might be responsible. Although the two exposure conditions, total-exhaust-exposure and filtered-exhaust-exposure had almost the same affect, the latter had a more severe impact on pups. When comparing pups from fetal-period-filtered-exposed group (*Filtered-C-C*) to those of the fetal-period-total-exposed group (*Total-C-C*), the thymus weights were significantly lower in *Filtered-C-C* than in the *Control* on postnatal day 96. In pups from the suckling-period-exposed group (*C-Filtered-C*), the thymus weights on days 23, 49 and 96 were lower than those in the *Control.* There were no significant differences between the thymus weight of *Filtered-C-C* and *Total-C-C.* Filtered exhaust contained the remainder of diesel exhaust after filtration, such as gases and particles measuring less than 0.05 μm. The gaseous phase of diesel engine exhaust includes several agents that may affect immune functions, such as nitrogen dioxide and nitrogen oxide \[[@B46]-[@B48]\]. Recently the toxicity of ultrafine particles less than 0.1 μm has been reported, such as the direct invasion of ultrafine particles through the lung, the translocation of deposited ultrafine particles to extrapulmonary tissues, the impact of co-pollutants adsorbed to ultrafine particles \[[@B52]-[@B54]\] . It can not be negligible that ultrafine particles transferred from the mother to the fetus or inhaled act as immunotoxicants. Further studies are needed to show the mechanism that is responsible for susceptibility to allergies in rats exposed to diesel exhaust during the critical period of immune system development.

The yearly average suspended particulate matter (SPM) concentration in a specified area at the motor vehicle exhaust monitoring station of motor vehicle NOx during 1999 was 0.045 mg/m^3^\[[@B51]\]. Assuming the proportion of diesel exhaust particles in SPM around the roads in large cities to be 40%, the concentration of the diesel exhaust particles used in this experiment is about 90 times the concentration occurring around the roads. The rats had been exposed to diesel exhaust for 16 days during their fetal life, which is more than two thirds of the gestation period. Taking the longer gestation period of human beings, a longer time of exposure per day and a larger volume of breathing into consideration, the inhalation dose used in the present study was not far from reality.

The intranasal administration is a more reasonable simulation of the natural route of pollen exposure. It is also useful to estimate the amount of pollen grains being administered. In the preliminary studies, intranasal administration was not useful for elevating IgE in Fischer rats. The major purposes of the study were to show that environmental factors could change the sensibility to pollen besides genetic factors and to show the influence of diesel exhaust inhalation on the fetus and neonate. Antibody responses against Japanese cedar pollen differed among strains of rats, with Brown Norway (BN) rats being sensitive, Fischer rats moderately sensitive and Wistar rats not sensitive \[[@B36]\]. The average litter size in BN rats is 3 to 4, and that of Fischer rats is 8 to 10. For these reasons, intraperitoneal injection was administered in Fischer rats in the present study. Even if alternative routes of pollen grain administration were used, the predictability of such methods generally used in scientific research field is well acceptable to mimic and study human allergic reaction in a rat model.

Competing interests
===================

none declared

Authors\' contributions
=======================

NW designed the study, carried out the immunoassays and drafted the manuscript. MO carried out the regulation of exposure conditions in the inhalation chambers and participated in the immunoassays. All authors read and approved the final draft.

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

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Acknowledgements
================

This work was supported by CREST of JST (Japan Science and Technology Corporation).